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Diverse effects of parenteral arginine on systemic and local oxidanteantioxidant homeostasis and nitrosative stress in rats with subacute peritonitis Ya-Hui Chen, PhD,a,b Chien-Hsing Lee, MD, PhD,c,d Chien-Chou Hsiao, MD, MS,e Li-Sung Hsu, PhD,b and Hui-Chen Lo, PhDf,* a
Department of Medical Education and Research, Changhua Christian Hospital, Changhua, Taiwan, Republic of China Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung, Taiwan, Republic of China c Division of Pediatric Surgery, Changhua Christian Hospital, Changhua, Taiwan, Republic of China d Graduate Institute of Medical Sciences, Chang Jung Christian University, Tainan, Taiwan, Republic of China e Department of Pediatrics and Team of Clinical Nutrition Support Service, Changhua Christian Hospital, Changhua, Taiwan, Republic of China f Department of Nutritional Science, Fu Jen Catholic University, New Taipei City, Taiwan, Republic of China b
article info
abstract
Article history:
Background: The beneficial effects of arginine on oxidative stress have been previously re-
Received 30 May 2013
ported; however, excess production of nitric oxide, an arginine metabolite, may cause
Received in revised form
hemodynamic instability and inflammatory response. Previous studies have demonstrated
8 September 2013
that parenteral arginine levels at 2%e4% of total calories may alleviate inflammation and
Accepted 3 October 2013
enhance immunity, whereas greater than 6% of total calories may have adverse effects in
Available online 9 October 2013
rats with subacute peritonitis. Herein, we investigated the effects of parenteral arginine dose on lipid peroxidation (thiobarbituric acid reactive substances, TBARS) and antioxidant
Keywords:
enzyme activities in the plasma and organs.
Peritonitis
Materials and methods: Male Wistar rats with cecal punctureeinduced subacute peritonitis
Arginine
were infused with parenteral nutrition solutions containing 1.61% (CP group), 2.85% (LA
Nitric oxide
group), 4.08% (MA group), and 6.54% (HA group) of total calories as arginine for 7 d. Healthy,
Lipid peroxidation
orally fed rats (NC group) were used as references.
Antioxidant enzymes
Results: Subacute peritonitis significantly elevated the levels of nitrate, nitrite and TBARS in
Nitrotyrosine
the plasma and decreased glutathione peroxidase activity in the kidneys. These changes
Subacute peritonitis
were significantly reversed in the MA and HA groups. The MA and HA groups had signif-
Parenteral nutrition
icantly increased nitrotyrosine levels in the plasma. The LA, MA, and HA groups had significantly increased glutathione peroxidase activity in the plasma, cytochrome P450 levels in the liver, and nitrotyrosine levels in the heart and had significantly decreased TBARS levels in the kidneys compared with the CP group. Conclusions: Our results suggest that parenteral arginine at a dose less than 4% of total calories may attenuate lipid peroxidation and increase antioxidant enzyme activities without leading to nitrosative stress in subacute peritonitis. ª 2014 Elsevier Inc. All rights reserved.
* Corresponding author. Department of Nutritional Science, Fu Jen Catholic University, #510 Zhongzheng Road, Xinzhuang District, New Taipei City, 24205, Taiwan, Republic of China. Tel.: þ886 2 2905 2547; fax: þ886 2 2902 1215. E-mail address: [email protected] (H.-C. Lo). 0022-4804/$ e see front matter ª 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2013.10.002
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1.
Introduction
Peritonitis remains a life-threatening disease, particularly when multiorgan dysfunction develops. It has been hypothesized that the pathogenesis of multiple organ failure in peritonitis is a result of the overproduction of nitric oxide (NO), reactive oxygen species (ROS), and inflammatory cytokines [1e4]. Recent evidence showed that peritonitic patients had increased circulating malondialdehyde and nitrate, that is, the derivatives of lipid peroxidation and NO, and decreased thiols in the ascetic fluid [4]. In peritonitic animals, the levels of antioxidants, such as ascorbic acid (vitamin C) and glutathione (GSH), and the activities of antioxidant enzymes, such as superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx), were significantly decreased in the blood, liver, and lungs [5]. These findings indicated that peritonitis is associated with elevated oxidative stress and weakened antioxidant defense system; thus, a disturbed oxidanteantioxidant homeostasis plays a certain role in the development of multiple organ failure [6]. The peritonitis-induced metabolic changes may result in muscle loss, organ dysfunction, and immune dysregulation [7]. To improve hypercatabolic status, parenteral nutrition has been introduced to patients with peritonitis [8]. However, the prolonged administration of a parenteral solution increases the risk of cholestasis, hepatic steatosis, and liver dysfunction [9] for it may elevate oxidative stress and eliminate antioxidant defenses [10]. Several studies indicated that arginine supplementation may decrease oxidative stress in rats with exhaustive exercise [11], diabetes [12], ulcerative colitis [13], and parenteral feedingeinduced hepatic steatosis [14] and in patients with myocardial ischemic disorders [15], as evidenced by the decreased malondialdehyde levels, the increased GSH levels, and the elevated SOD and catalase activities in the plasma and different organs. These beneficial effects of arginine are closely related to the arginine metabolite NO, a free radical scavenger that may inhibit the activity of pro-oxidant enzymes. Nevertheless, Dioguardi [16] indicated that with greater amounts of arginine introduced, more arginine is destroyed, and impaired NO production eventually occurs in patients with myocardial infarction. This paradox may be related to the production of endogenous inhibitor of nitric oxide synthase, that is, asymmetric dimethylarginine (ADMA) [17]. Using a rat model with subacute peritonitis, that is, nonlethal inflammation, our previous study revealed that the beneficial effects of arginine are dose limited. For example, parenteral arginine may facilitate ureagenesis and proline conversion without resulting in elevated NO levels [18] and may alter leukocytic and splenocytic immunity in an inverted U-shaped dose-dependent manner [19]. These results suggest that arginine replenishment should be carefully evaluated in patients with inflammation, as the arginine-derived metabolite NO may react with superoxide anion to form the toxic compound peroxynitrite [20]. To date, it is still not known whether this dose-dependent effect of parenteral arginine supplementation is associated with changes in oxidanteantioxidant homeostasis. Therefore, in the present study, we investigated the effects of parenteral arginine
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dosage on the levels of lipid peroxidation, antioxidants, antioxidant enzyme activity, and nitrotyrosine, an indirect marker of peroxynitrite [2], in the plasma and peritonitisaffected target organs, including the heart, lungs, liver, and kidneys of rats with subacute peritonitis.
2.
Methods and materials
2.1.
Experimental protocol
The animal facilities and protocols were reviewed and approved by the Institutional Animal Care and Use Committee in Changhua Christian Hospital, Changhua, Taiwan. Thirty male Wistar rats (National Taiwan University, Taipei, Taiwan) initially weighing approximately 250 g were housed in individual stainless steel cages with free access to water and rat chow (1320 Rat and Mouse Maintenance diet; Altromin GmbH, Germany) in a room maintained at 22 C on a 12:12-h lightedark cycle. After being acclimated for 5 d, six rats maintained on chow diet, water, and nonsurgical procedures were used as normal controls (NC group). The other 24 rats were fasted overnight and anesthetized with intramuscular injection of 100 mg of ketamine and 10 mg of xylazine per kilogram of body weight before surgery (day 0). Subsequently, two surgeries were performed as follows: the placement of catheters in the superior vena cava by way of the external jugular vein for the infusion of parenteral nutrition solutions, and a midline incision in the abdomen with two punctures through both sides of the exposed cecum, as described in our previous study [18]. When making the two cecal punctures, a small amount of fecal material of the size of a rice grain was extruded from each of the puncture sites. After replacing the cecum and fecal material into the abdominal cavity, the abdomen was closed with 3-0 chromic gut sutures for the muscle layer and surgical staples for the skin. After the surgeries, the animals were infused with four different types of parenteral solutions providing equal amounts of calories and 3.97 (standard parenteral solution, CP group), 7.01 (low dose, LA group), 10.58 (medium dose, MA group), or 16.15 (high dose, HA group) grams of arginine per liter of solution for 7 d. The amounts of arginine supplementation accounted for 1.61%, 2.85%, 4.08%, and 6.54% of the total calories in the CP, LA, MA, and HA groups, respectively. During the experimental period, water was provided ad libitum, and the parenteral solutions were infused gradually from 26 mL on day 0 to 62 mL (234 kcal/kg/d) on day 1 through day 6, which is adequate for healthy rats of this size. On day 7, the rats were euthanized under anesthesia via intramuscular injections of 100 mg of ketamine and 10 mg of xylazine per kilogram of body weight. Blood was collected by cardiac puncture, and serum and plasma were obtained for different assays. The liver, lung, heart, and kidneys were dissected and stored at 80 C until further assay.
2.2.
Analytical measurements
To assess the nutritional status and clinical biochemical changes in rats, the serum concentrations of glucose, albumin,
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triglyceride, blood urea nitrogen (BUN), creatinine, glutamic oxaloacetic transaminase (GOT), and glutamic pyruvic transaminase (GPT) were measured by an automatic analyzer (Hitachi 747; Tokyo, Japan). To evaluate the nitrosative status, the plasma concentrations and organ contents of nitrite and nitrate (NOx), which indirectly represent NO, were determined by a commercial colorimetric kit (Cayman Chemical, Ann Arbor, MI). In addition, nitrotyrosine, a specific marker for protein modification by NO-derived oxidants, was measured using an enzyme-linked immunosorbent assay kit (OXIS International, Inc, Oxis Research, Portland, OR). Thiobarbituric acid reactive substances (TBARS), the products of lipid peroxidation as the representative indices of oxidative stress, were determined in the plasma and organs as described by Ohkawa et al. [21]. To determine the levels of sulfhydryl groups, that is, thiol groups, 0.2 mol/L of Tris buffer (pH 8.2) with or without 0.01 mol/L of 5,50 -dithio-bis(2nitrobeznoic acid) was added to the plasma and organ homogenates (3:1 vol/vol). After incubation for 30 min and centrifugation at 3000g for 30 min, the levels of the thiol groups in the supernatants were determined at 412 nm according to the method described by Sedlak and Lindsay [22]. The activities of antioxidant enzymes, that is, SOD, catalase, and GPx, were measured in the plasma and organ homogenates after individual correction of protein concentrations. Protein levels of the plasma and organ homogenates were determined using a commercially available kit (Bicinchoninic Acid Protein Assay; Pierce Chemical Co, Rockford, IL). SOD activity was evaluated according to the inhibition of the pyrogallol autoxidation rate and was expressed in pyrogallol units (U/mg protein) using the method described by Marklund and Marklund [23]. The activity of catalase was determined spectrophotometrically by monitoring the rate of hydrogen peroxide (H2O2) decomposition at 240 nm at 30-s intervals for 3 min using an extinction coefficient of 43.6/(M cm) for H2O2, according to the method described by Aebi [24]. The activity of catalase was expressed as nanomoles of H2O2 decomposition per minute per milligram of protein. The activity of GPx was determined by the method modified from Paglia and Valentine [25] and expressed as nanomoles of reduced nicotinamide adenine dinucleotide phosphate (NADPH) to oxidized nicotinamide adenine dinucleotide phosphate (NADP) per minute per milligram of protein using an extinction coefficient of 6.22 106 for NADPH. The content of cytochrome P450 in the liver microsomes was measured according to the method of Omura and Sato [26]. Microsomes of liver homogenates were extracted and precipitated. The content of cytochrome P450 was determined from the different carbon monoxide spectra of dithionite-reduced samples, assuming a value of 91/(mM cm) for the molar extinction coefficient between 450 and 490 nm, and was expressed as picomoles of per milligram of microsomal protein.
2.3.
Statistical analysis
All treatment groups were compared with a one-way analysis of variance (ANOVA) using the SAS general linear models program. Values are reported as the means standard error of the mean. Group means were considered to be significantly
different at P < 0.05, as determined by the post hoc analysis of protective least-significant difference, in which the ANOVA indicated an overall significant treatment effect at P < 0.05.
3.
Results
3.1.
Parenteral solution infusion and body weights
The amounts of parenteral solution infusion were not significantly different among the four parenterally fed groups with subacute peritonitis during the experimental period (62.4e64.8 kcal/d from day 1 to day 7). After 7 d, the healthy, orally fed rats, that is, the NC group, had a significantly increased body weight (60.71 4.91 g) compared with the peritonitic rats with parenteral nutrition, that is, the CP (0.83 2.68 g), LA (3.00 5.15 g), MA (4.50 3.79 g), and HA (0.17 2.71 g) groups. The final sample size was six rats per group with a 100% survival rate.
3.2.
Clinical biochemical values
Subacute peritonitis was the major factor to alter the clinical biochemical values. Serum concentrations of glucose, albumin, BUN, creatinine, and GPT were significantly decreased in the CP group (i.e., 125.7 4.7 g/100 mL, 32.3 0.7 g/L, 4.64 0.14 mmol/L, 15.9 1.8 mmol/L, and 27.8 4.3 U/L, respectively) compared with the NC group (239.5 3.4 g/ 100 mL, 41.0 1.5 g/L, 8.90 0.50 mmol/L, 33.6 2.7 mmol/L, and 57.3 5.9 U/L, respectively). In contrast, serum concentrations of GOT were significantly increased in the CP group (143.2 9.6 U/L) compared with the NC group (79.0 3.2 U/L). Parenteral arginine did not have a significant impact on these clinical biochemical indices.
3.3.
Relative weights of organs
The relative weights, that is, organ weights divided by body weight on day 7, of the liver, lungs, heart, and kidneys are listed in Tables 2e5, respectively. Peritonitic rats with parenteral nutrition had significantly increased relative weights of the lungs compared with healthy, orally fed rats (P < 0.05). There were no significant differences in the relative weights of the liver, heart, and kidneys among groups.
3.4. Indices of nitrosative and oxidative stress and antioxidant status in the plasma and organs 3.4.1.
Plasma
The levels of NOx, nitrotyrosine, lipid peroxidation products (i.e., TBARS), and antioxidants (i.e., thiol groups), as well as the activities of antioxidant enzymes (i.e., SOD, GPx, and catalase) in the plasma, are shown in Table 1. Parenterally fed rats with subacute peritonitis, that is, the CP, LA, MA, and HA groups, had significantly increased the levels of NOx and TBARS and the CP, LA, and MA groups had significantly decreased the levels of nitrotyrosine and thiol groups in the plasma, compared with healthy, orally fed rats, that is, the NC group (P < 0.05). Peritonitic rats supplemented with medium and high doses of parenteral arginine had significantly
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Table 1 e The levels of NOx, nitrotyrosine, lipid peroxidation, and antioxidants, as well as the activities of antioxidant enzymes in the plasma. Group
NC
NOx, mmol/L Nitrotyrosine, nmol/L TBARS, nmol/mL Thiol group, nmol/mL SOD, U/mg protein GPx, nmol NADPH/min/mg protein Catalase, nmol H2O2/min/mg protein
12.3 8.29 3.64 217.2 310.5 4.01 1.37
CP 0.6 1.15 0.28 8.9 2.6 0.36 0.13
71.8 0.36 7.03 80.3 310.9 0.84 1.17
LA *
4.7 0.13* 0.12* 5.1* 13.4 0.01* 0.05
64.9 1.30 5.87 91.2 435.0 1.70 1.08
MA *
7.3 0.15* 0.53* 8.8* ,y 15.8* *,y 0.25 0.06
49.4 3.81 5.83 65.8 320.9 1.90 1.26
HA *,y
6.0 ,y 0.39* *,y 0.17 5.2* 6.4 ,y 0.13* 0.10
42.6 16.35 4.97 76.6 333.0 3.61 1.10
,y
4.1* ,y 2.89* *,y 0.55 4.8* 6.2 y 0.36 0.02
Values are represented as the means standard error of the mean; n ¼ 6 for each group. * Values are significantly different from the NC group at P < 0.05 by a one-way ANOVA. y Values are significantly different from the CP group at P < 0.05 by a one-way ANOVA.
differences in the contents of TBARS and thiol groups and the activities of SOD, GPx, or catalase in the lungs among groups.
reversed changes in plasma NOx, nitrotyrosine, and TBARS. Parenterally fed rats with subacute peritonitis had significantly decreased plasma GPx activities, and the low, medium, and high doses of parenteral arginine supplementation significantly attenuated these decreases. In addition, a low dose of arginine supplementation significantly increased plasma SOD activities in peritonitic rats. Neither peritonitis nor arginine supplementation had a significant impact on plasma catalase activity.
3.4.2.
3.4.4.
Liver
The NOx and nitrotyrosine contents, as well as the catalase activity, were significantly increased, whereas the thiol group contents and the GPx activity were significantly decreased in the liver in the CP, LA, MA, and HA groups compared with the NC group (Table 2). The LA, MA, and HA groups had 2-, 2-, and 4-fold increases in cytochrome P450 content in the liver compared with the NC and CP groups. There were no significant differences in the contents of TBARS in the liver among groups.
3.4.5. 3.4.3.
Heart
The concentrations of nitrotyrosine and TBARS and the activities of SOD, GPx, and catalase in the heart were significantly decreased in the CP group compared with the NC group (Table 4). The LA group had significantly increased the contents of NOx and TBARS, whereas the MA group had significantly increased NOx contents in the heart compared with the CP group. In addition, the peritonitis-induced decreases in the contents of nitrotyrosine were significantly elevated by parenteral arginine supplementation, as shown by the 4-, 4-, and 2-fold increases in the LA, MA, and HA groups, respectively, compared with the CP group. There were no significant differences in the contents of thiol groups in the heart among groups. In addition, parenteral arginine supplementation had no significant impact on the activities of SOD, GPx, or catalase in the hearts of rats with subacute peritonitis.
Kidneys
The contents of NOx and thiol groups were significantly increased, and the activity of GPx was significantly decreased in the kidneys of the CP group compared with the NC group (Table 5). The contents of nitrotyrosine were significantly increased in the LA and MA groups compared with the NC and CP groups. Parenteral arginine significantly decreased the
Lungs
The concentrations of NOx and nitrotyrosine in the lungs were significantly increased in the CP group compared with the NC group (Table 3). Peritonitic animals supplemented with a medium dose of parenteral arginine had a significantly decreased NOx content in the lungs. However, there were no significant
Table 2 e The relative weights, levels of NOx, nitrotyrosine, lipid peroxidation, and antioxidants, and activities of antioxidant enzymes in the liver. Group Relative weight, g/kg body weight NOx, mmol/g tissue Nitrotyrosine, pmol/mg protein TBARS, nmol/g tissue Thiol group, nmol/g tissue SOD, U/mg protein GPx, nmol NADPH/min/mg protein Catalase, nmol H2O2/min/mg protein Cytochrome P450, pmol/mg protein
NC 51.29 255 3.28 514.3 20.90 141.9 546.8 43.16 8.31
CP 2.90 16 0.33 30.0 0.71 8.5 46.2 10.8 2.02
46.91 366 5.28 492.1 17.29 186.4 297.3 309.1 9.33
LA 2.18 11* 0.22* 36.4 0.76* 11.7 26.3* 31.8* 1.17
47.40 383 5.50 524.8 17.44 253.8 336.6 242.4 17.07
Values are represented as the means standard error of the mean; n ¼ 6 for each group. * Values are significantly different from the NC group at P < 0.05 by a one-way ANOVA. y Values are significantly different from the CP group at P < 0.05 by a one-way ANOVA.
2.07 24* 0.61* 35.4 0.48* 20.8* 19.3* 23.4* 2.58*,y
MA 42.95 392 6.07 483.2 17.11 222.9 321.5 278.6 17.97
2.73 12* 0.49* 46.0 0.73* 27.0* 11.7* 21.2* 2.30*,y
HA 46.82 351 6.54 502.8 18.11 196.2 281.3 402.4 41.10
0.74 11* 0.70* 45.3 0.34* 18.4 9.5* 53.8* 3.29*,y
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Table 3 e The relative weights, levels of NOx, nitrotyrosine, lipid peroxidation, and antioxidants, and activities of antioxidant enzymes in the lung. Group
NC
Relative weights, g/kg body weight NOx, mmol/g tissue Nitrotyrosine, pmol/mg protein TBARS, nmol/g tissue Thiol group, nmol/g tissue SOD, U/mg protein GPx, nmol NADPH/min/mg protein Catalase, nmol H2O2/min/mg protein
6.37 299 17.5 319.5 8.20 74.26 538.9 1075
0.26 44 2.0 32.9 0.81 10.31 45.8 54
CP 8.69 491 30.2 394.9 7.32 92.06 596.7 1067
LA 0.32 35* 2.7* 21.7 0.40 7.02 46.4 30
*
9.14 419 28.8 389.2 7.53 84.78 551.5 981
MA *
0.35 47* 3.2* 27.8 0.33 5.81 53.2 62
8.18 366 33.8 364.9 7.78 90.72 590.9 1010
0.38 30y 2.9* 24.9 0.69 3.75 51.3 56
HA *
8.60 435 34.2 347.2 8.19 99.03 550.0 1021
0.18* 20* 3.6* 12.6 0.16 5.80 38.5 33
Values are represented as the means standard error of the mean; n ¼ 6 for each group. * Values are significantly different from the NC group at P < 0.05 by a one-way ANOVA. y Values are significantly different from the CP groups at P < 0.05 by a one-way ANOVA.
contents of TBARS and thiol groups in the kidneys in a dosedependent manner. In addition, the peritonitis-induced decreases in the GPx activity of the kidneys were significantly increased in rats supplemented with medium and high doses of parenteral arginine. There were no significant differences in the activities of SOD and catalase in the kidneys among groups.
4.
Discussion
It has been demonstrated that activated leukocytes and neutrophils may produce excessive ROS and NO to ignite inflammatory response in trauma and sepsis, which, in turn, lead to organelle dysfunction and multiple organ failure [6]. Conditions of arginine deficiency worsen protein metabolism, microcirculation, immune function, and antioxidant status in sepsis [27]. However, arguments have been raised against arginine supplementation concerning the toxicity of excess NO and hemodynamic instability. Previous studies have shown that parenteral arginine at a dose of 6.54% of the total calories may not elevate circulating NOx levels [18] and that 2%e4% of total calories may enhance the immunities of peripheral blood cells and splenocytes in rats with subacute peritonitis [19]. In this study, we showed that parenteral arginine may attenuate lipid peroxidation and elevate GPx activity, although potentially increasing the NOx and
nitrotyrosine contents in the plasma and in certain organs in rats with subacute peritonitis. In the present study, subacute peritonitic rats with parenteral feeding had significantly lower body weight gains; indices of nutrition status, such as serum glucose, albumin, and triglyceride; and serum BUN and creatinine levels compared with healthy rats with oral feeding. Our previous study showed that rats with subacute peritonitis have significantly decreased serum insulin-like growth factor (IGF)-I, a commonly used anabolic hormone to assess nutrition status that has an insulin-like action and a short half-life (10e20 min) [18]. These results implied that rats with subacute peritonitis may be in some degree of undernutrition. When supplemented with a low dose of parenteral arginine, the peritonitis-induced decrease in serum IGF-I was attenuated [18], but body weight, serum albumin, and serum insulin were not significantly affected. The relative lung weights (Table 3), serum GOT, and cytochrome P450 content in the liver (Table 2) were significantly increased, and the serum GPT was significantly decreased by parenteral arginine. These changes suggested that although parenteral arginine supplementation may not improve whole body catabolism, it still has certain effects on the lung, heart, liver, and kidneys in rats with subacute peritonitis. Evidence has shown that patients with septic shock and secondary organ dysfunction showed significantly decreased plasma antioxidant status, increased plasma lipid peroxidation, and elevated urinary nitrite excretion [6,28]. In subacute
Table 4 e The relative weights, levels of NOx, nitrotyrosine, lipid peroxidation, and antioxidants, and activities of antioxidant enzymes in the heart. Group Relative weights, g/kg body weight NOx, mmol/g tissue Nitrotyrosine, pmol/mg protein TBARS, nmol/g tissue Thiol group, nmol/g tissue SOD, U/mg protein GPx, nmol NADPH/min/mg protein Catalase, nmol H2O2/min/mg protein
NC 3.60 243 50.46 597.2 13.04 191.0 539.4 1011
CP 0.11 22 4.83 48.2 0.24 18.1 25.6 40
3.96 207 26.74 490.1 13.15 142.1 376.8 689
LA 0.26 18 7.56* 28.0* 0.42 3.3* 16.7* 24*
4.18 423 110.35 585.1 11.62 128.9 399.9 722
Values are represented as the means standard error of the mean; n ¼ 6 for each group. * Values are significantly different from the NC group at P < 0.05 by a one-way ANOVA. y Values are significantly different from the CP group at P < 0.05 by a one-way ANOVA.
0.14 ,y 13* ,y 15.19* y 24.8 0.67 6.6* 27.5* 17*
MA 4.32 292 103.88 547.9 12.21 127.0 385.4 756
0.35 22y ,y 16.94* 24.4 0.76 4.8* 20.6* 33*
HA 4.01 207 56.92 507.6 12.14 147.9 394.7 780
0.12 11 ,y 8.39* * 17.3 0.52 6.1* 13.0* 32*
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Table 5 e The relative weights, levels of NOx, nitrotyrosine, lipid peroxidation, and antioxidants, and activities of antioxidant enzymes in the kidneys. Group Relative weight, g/kg body weight NOx, mmol/g tissue Nitrotyrosine, pmol/mg protein TBARS, nmol/g tissue Thiol group, nmol/g tissue SOD, U/mg protein GPx, nmol NADPH/min/mg protein Catalase, nmol H2O2/min/mg protein
NC 10.97 139 87.6 419.7 13.01 270.6 1112 566.8
CP 0.54 5 6.0 9.0 1.19 9.6 117 44.1
10.34 250 148.6 383.2 15.08 265.7 902 652.7
LA 0.16 27* 18.7 8.7 0.46* 8.6 42* 28.5
10.98 138 347.7 290.5 13.12 294.1 1022 660.8
0.37 18 ,y 39.5* *,y 22.8 0.34y 8.4 63 24.3
MA 10.44 251 465.2 265.2 12.77 281.4 1157 591.2
0.59 35* ,y 44.9* *,y 36.2 0.29y 10.8 45y 57.9
HA 11.06 264 110.8 238.1 11.94 290.8 1104 638.0
0.13 54* 9.9 ,y 30.8* y 0.51 10.3 51y 29.4
Values are represented as the means standard error of the mean; n ¼ 6 for each group. * Values are significantly different from the NC group at P < 0.05 by a one-way ANOVA. y Values are significantly different from the CP groups at P < 0.05 by a one-way ANOVA.
peritonitic rats with parenteral nutrition, the levels of NOx and TBARS in the plasma were significantly increased, whereas nitrotyrosine and thiol group levels in the plasma, the physiological free radical scavengers, and GPx activities were significantly decreased (Table 1). These changes revealed that these rats had a systemic imbalance in oxidanteantioxidant homeostasis. A similar imbalance was found in the organs of these rats, such as the significant increases in NOx levels in the liver, lungs, and kidneys and the significant decreases in thiol group levels in the liver, GPx activity in the liver, heart, and kidneys, and SOD and catalase activities in the heart. These results indicated that subacute peritonitic rats with parenteral nutrition were under conditions of systemic and local oxidative stress and antioxidant defense defects. The beneficial effects of arginine on oxidanteantioxidant homeostasis have been observed in inflammation and sepsis [13e15]. It has been proposed that the “arginine paradox” may impair NO production because of the endogenous inhibitor of nitric oxide synthase, that is, ADMA [16]. In the present study, parenteral arginine supplementation at 4%e6%, but not at 2%, of the total calories significantly alleviated the increases in the levels of NOx and TBARS in the plasma in the peritonitic rats. These results suggest that parenteral arginine at 4%e6% of the total calories may represent a condition of the arginine paradox; however, we did not measure the levels of ADMA in the present study to prove it. It is also possible that argininederived NO reacts with superoxide to decrease the production of lipid peroxidation, as shown in exhaustively exercised rats fed with a diet supplemented with 2% arginine [29]. Arginine supplementation may reduce the exercise-induced oxidanteantioxidant imbalance and this modulation is likely mediated through the arginineeNO pathways [30]. However, the increased accumulation of nitrotyrosine, a product generated by NO and superoxide (O 2 ), in the kidneys was found to be mainly because of the oxidation of NO by ROS in rats with GSH depletion-induced hypertension [31]. In this study, subacute peritonitic rats had significantly decreased plasma thiol levels and GPx activity, and parenteral arginine significantly elevated the plasma nitrotyrosine levels and GPx activities, whereas decreasing the levels of NOx and TBARS in the plasma. These results support the concept that the arginine-derived NO may react with superoxides to decrease the production of lipid peroxidation in subacute peritonitic rats with parenteral arginine supplementation. However, the
arginine-induced increase in plasma nitrotyrosine may still be able to cause adverse effects. In septic rats, Mu¨ller et al. [32] found that plasma NOx concentrations were negatively correlated with the total cytochrome P450 content in the liver microsomes. In this study, peritonitic rats supplemented with parenteral arginine at 2.85%, 4.08%, and 6.54% of total calories had 2-, 2-, and 4-fold increases in the total cytochrome P450 content in the liver and 0.9-, 0.7-, and 0.6-fold increases in plasma NOx levels compared with those with parenteral arginine as 1.61% of total calories. We confirmed that there is a negative relationship between the plasma NOx and total cytochrome P450 content in the liver of subacute peritonitic rats with parenteral arginine supplementation. In addition, we found that greater than 6% of total calories as parenteral arginine may result in a greater increase in the oxidation of organic substances in the liver. Oxidanteantioxidant homeostasis in the body is sensitively controlled by two antioxidant defense systems, that is, nonenzymatic antioxidants, including vitamins, minerals, cofactors, and thiols, and enzymatic antioxidants, including SOD, catalase, GPx, and GSH reductase. In conditions of trauma, burn, or sepsis, oxygen-derived free radicals are excessively produced, activating SOD to excessively catalyze the conversion of superoxide into H2O2. Then, H2O2 is further detoxified by catalase and GPx [27,33]. Warner et al. [34] found that septic patients had significantly elevated total plasma SOD activity compared with healthy controls, whereas septic survivors had significantly lower total plasma SOD and catalase activities than septic nonsurvivors. In addition, the elevated activities of serum SOD and GPx and the increased extent of lipid peroxidation were observed in septic neonates [35]. In the present study, subacute peritonitic rats did not show significant changes in the plasma activities of SOD and catalase but instead had significantly decreased plasma GPx activity and increased plasma TBARS levels. These inconsistent changes in antioxidant enzymes might be because of differing severities and phases of sepsis, as suggested by Andrades et al. [36]. These changes in antioxidant enzymes seem to differ between nonlethal and lethal sepsis. Our findings also showed that subacute peritonitic rats supplemented with parenteral arginine may have elevated plasma SOD and GPx activities and decreased plasma lipid peroxidation, which suggest that parenteral arginine may partially alleviate systemic oxidative stress.
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A recent study reported that oxidanteantioxidant homeostasis may be varied in the circulation and organs, depending on the physiological functions and the degree of oxidative damage [36]. For example, catalase activity was decreased in the livers of septic rats with cecal ligation and punctures [37]. In addition, TBARS were positively correlated with lung and renal damage, whereas oxidative damage was only correlated with an increase in the ratio of SOD to catalase in the lungs, but not in kidneys, of the lethal septic rats [38]. However, the increases in the ratio of SOD to catalase in the heart, liver, lung, and kidneys of the nonlethal septic rats were much lower than those of the lethal septic rats [36]. In subacute peritonitic rats, a nonlethal septic rat model, we found that GPx activity was decreased, catalase activity and the cytochrome P450 content were increased, and the content of TBARS was not changed in the liver. These results suggest that an oxidanteantioxidant balance in the liver may be achieved. In the heart, the activities of SOD, GPx, and catalase were significantly decreased. Accompanied with the increased serum GOT levels, we speculated that these subacute peritonitic rats might exhibit a certain degree of heart damage; however, we did not measure heart function to confirm it. Herein, we showed different changes in the activities of antioxidant enzymes, revealing that oxidanteantioxidant homeostasis may vary across different organs. In addition, parenteral arginine supplementation at a dose greater than 4% of the total calories can increase the GPx activity in the kidneys. Whether this effect is associated with the physiological function of the kidneys in arginine metabolism needs further investigation. The present study has several limitations. First, there were no sham-operated and parenteral-fed rats as controls to distinguish between the individual effects of parenteral feeding and subacute peritonitis on the oxidative stress and antioxidant defense system. Therefore, the results of the CP group represented the combined effects of parenteral feeding and subacute peritonitis. Second, we only obtained samples on day 7 to investigate the long-term effects of parenteral arginine supplementation. Thus, we might have overlooked more immediate effects of parenteral arginine. Third, we did not measure the protein or messenger RNA levels of these antioxidant enzymes. It might be possible to demonstrate the effects of parenteral arginine on the molecular levels of these enzymes. Finally, more assays should be performed; for example, blood gas assays for lung function and cardiac indices for heart performance would facilitate the investigation of whether parenteral arginine supplementation improves organ function via the changes in the oxidative stress and antioxidant defense systems.
5.
Conclusions
The results of this study revealed that subacute peritonitis significantly increased systemic and local oxidative and nitrosative stresses and diminished the antioxidant defense system. In addition, parenteral arginine at a dose of 4%e6% of the total calories may attenuate lipid peroxidation and elevate antioxidant enzyme activity and nitrosative stress.
Therefore, parenteral arginine supplementation may maintain oxidanteantioxidant homeostasis in a dose- and organdependent manner. To sum up, parenteral arginine at a dose less than 4% of total calories may attenuate lipid peroxidation and increase antioxidant enzyme activities through L-arginineeNO pathways without leading to nitrosative stress in subacute peritonitis.
Acknowledgment This work was supported by the National Science Council of the Republic of China under the grant number NSC-95-2320-B309-001 and by Changhua Christian Hospital under the grant number CCH-92106.
references
[1] Kim YM, Hong SJ, Billiar TR, Simmons RL. Counterprotective effect of erythrocytes in experimental bacterial peritonitis is due to scavenging of nitric oxide and reactive oxygen intermediates. Infect Immun 1996;64:3074. [2] Xie YW, Wolin MS. Role of nitric oxide and its interaction with superoxide in the suppression of cardiac muscle mitochondrial respiration. Involvement in response to hypoxia/reoxygenation. Circulation 1996;94:2580. [3] Cuzzocrea S, Zingarelli B, Costantino G, Sottile A, Teti D, Caputi AP. Protective effect of poly(ADP-ribose) synthetase inhibition on multiple organ failure after zymosan-induced peritonitis in the rat. Crit Care Med 1999;27:1517. [4] Natarajan SK, Mukhopadhya A, Ramachandran A, Amalanathan S, Kurian G, Balasubramanian KA. Spontaneous bacterial peritonitis results in oxidative and nitrosative stress in ascitic fluid. J Gastroenterol Hepatol 2007;22:177. [5] Demling R, Nayak U, Ikegami K, LaLonde C. Comparison between lung and liver lipid peroxidation and mortality after zymosan peritonitis in the rat. Shock 1994;2:222. [6] Vı´ctor VM, Espulgues JV, Herna´ndez-Mijares A, Rocha M. Oxidative stress and mitochondrial dysfunction in sepsis: a potential therapy with mitochondria-targeted antioxidants. Infect Disord Drug Targets 2009;9:376. [7] Michie HR. Metabolism of sepsis and multiple organ failure. World J Surg 1996;20:460. [8] Ikeda K, Kimura Y, Iwaya T, et al. Perioperative nutrition for gastrointestinal surgery. Nippon Geka Gakkai Zasshi 2004; 105:218. [9] Weinberger B, Watorek K, Strauss R, Witz G, Hiatt M, Hegyi T. Association of lipid peroxidation with hepatocellular injury in preterm infants. Crit Care 2002;6:521. [10] Cai W, Wu J, Hong L, Xu Y, Tang Q, Shi C. Oxidative injury and hepatocyte apoptosis in total parenteral nutrition-associated liver dysfunction. J Pediatr Surg 2006;41: 1663. [11] Huang CC, Lin TJ, Lu YF, Chen CC, Huang CY, Lin WT. Protective effects of L-arginine supplementation against exhaustive exercise-induced oxidative stress in young rat tissues. Chin J Physiol 2009;52:306. [12] Coronel I, Arellano-Mendoza MG, del Valle-Mondragon L, et al. L-arginine and antioxidant diet supplementation partially restores nitric oxide-dependent regulation of phenylephrine renal vasoconstriction in diabetics rats. J Ren Nutr 2010;20:158.
j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 7 ( 2 0 1 4 ) 1 3 4 e1 4 1
[13] Harisa GE, Abo-Salem OM, El-Sayed el-SM, Taha EI, ElHalawany N. L-arginine augments the antioxidant effect of garlic against acetic acid-induced ulcerative colitis in rats. Pak J Pharm Sci 2009;22:373. [14] Ilan E, Tirosh O, Madar Z. Triacylglycerol-mediated oxidative stress inhibits nitric oxide production in rat isolated hepatocytes. J Nutr 2005;135:2090. [15] Tripathi P, Misra MK. Therapeutic role of L-arginine on free radical scavenging system in ischemic heart diseases. Indian J Biochem Biophys 2009;46:498. [16] Dioguardi FS. To give or not to give? Lessons from the arginine paradox. J Nutrigenet Nutrigenomics 2011;4:90. [17] Bo¨ger RH. Asymmetric dimethylarginine, an endogenous inhibitor of nitric oxide synthase, explains the “l-arginine paradox” and acts as a novel cardiovascular risk factor. J Nutr 2004;134:2842S. [18] Lo HC, Wu SC, Li ML, Lee CH. Does pharmacological dose of parenteral arginine have beneficial effect in rats with subacute peritonitis? Pediatr Surg Int 2010;26:625. [19] Chen YH, Lee CH, Hsu LS, Hsiao CC, Lo HC. Appropriate dose of parenteral arginine enhances immunity of peripheral blood cells and splenocytes in rats with subacute peritonitis. JPEN J Parenter Enteral Nutr 2012;36:741. [20] Pacher P, Beckman JS, Liaudet L. Nitric oxide and peroxynitrite in health and disease. Physiol Rev 2007;87:315. [21] Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979;95:351. [22] Sedlak J, Lindsay RH. Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman’s reagent. Anal Biochem 1968;25:192. [23] Marklund S, Marklund G. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem 1974;47:469. [24] Aebi HE. Catalase. In: Bergmeyer HU, editor. Methods of enzymatic analysisvol. 3. Weinheim: VCH Verlag; 1987. p. 273e8. [25] Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 1979;70:158. [26] Omura T, Sato R. The carbon monoxide-binding pigment of liver microsomes. J Biol Chem 1964;239:2379.
141
[27] Luiking YC, Deutz NE. Exogenous arginine in sepsis. Crit Care Med 2007;35(9 Suppl):S557. [28] Goode HF, Cowley HC, Walker BE, Howdle PD, Webster NR. Decreased antioxidant status and increased lipid peroxidation in patients with septic shock and secondary organ dysfunction. Crit Care Med 1995;23:646. [29] Lin WT, Yang SC, Tsai SC, Huang CC, Lee NY. L-arginine attenuates xanthine oxidase and myeloperoxidase activities in hearts of rats during exhaustive exercise. Br J Nutr 2006;95:67. [30] Shan L, Wang B, Gao G, Cao W, Zhang Y. L-arginine supplementation improves anti-oxidant defenses through L-arginine/nitric oxide pathways in exercise rats. J Appl Physiol; 2013 [Epub ahead of print]. [31] Zhou XJ, Vaziri ND, Wang XQ, Silva FG, Laszik Z. Nitric oxide synthase expression in hypertension induced by inhibition of glutathione synthase. J Pharmacol Exp Ther 2002;300:762. [32] Mu¨ller CM, Scierka A, Stiller RL, et al. Nitric oxide mediates hepatic cytochrome P450 dysfunction induced by endotoxin. Anesthesiology 1996;84:1435. [33] Guemouri L, Artur Y, Herbeth B, Jeandel C, Cuny G, Siest G. Biological variability of superoxide dismutase, glutathione peroxidase, and catalase in blood. Clin Chem 1991;37:1932. [34] Warner A, Bencosme A, Healy D, Verme C. Prognostic role of antioxidant enzymes in sepsis: preliminary assessment. Clin Chem 1995;41(6 Pt 1):867. [35] Barbas C, Castro M, Bonet B, Viana M, Herrera E. Simultaneous determination of vitamins A and E in rat tissues by high-performance liquid chromatography. J Chromatogr A 1997;778:415. [36] Andrades M, Ritter C, Moreira JC, Dal-Pizzol F. Oxidative parameters differences during non-lethal and lethal sepsis development. J Surg Res 2005;125:68. [37] Llesuy S, Evelson P, Gonza´lez-Flecha B, et al. Oxidative stress in muscle and liver of rats with septic syndrome. Free Radic Biol Med 1994;16:445. [38] Andrades M, Ritter C, de Oliveira MR, Streck EL, Fonseca Moreira JC, Dal-Pizzol F. Antioxidant treatment reverses organ failure in rat model of sepsis: role of antioxidant enzymes imbalance, neutrophil infiltration, and oxidative stress. J Surg Res 2011;167:e307.
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Diverse effects of parenteral arginine on systemic and local oxidanteantioxidant homeostasis and nitrosative stress in rats with subacute peritonitis Ya-Hui Chen, PhD,a,b Chien-Hsing Lee, MD, PhD,c,d Chien-Chou Hsiao, MD, MS,e Li-Sung Hsu, PhD,b and Hui-Chen Lo, PhDf,* a
Department of Medical Education and Research, Changhua Christian Hospital, Changhua, Taiwan, Republic of China Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung, Taiwan, Republic of China c Division of Pediatric Surgery, Changhua Christian Hospital, Changhua, Taiwan, Republic of China d Graduate Institute of Medical Sciences, Chang Jung Christian University, Tainan, Taiwan, Republic of China e Department of Pediatrics and Team of Clinical Nutrition Support Service, Changhua Christian Hospital, Changhua, Taiwan, Republic of China f Department of Nutritional Science, Fu Jen Catholic University, New Taipei City, Taiwan, Republic of China b
article info
abstract
Article history:
Background: The beneficial effects of arginine on oxidative stress have been previously re-
Received 30 May 2013
ported; however, excess production of nitric oxide, an arginine metabolite, may cause
Received in revised form
hemodynamic instability and inflammatory response. Previous studies have demonstrated
8 September 2013
that parenteral arginine levels at 2%e4% of total calories may alleviate inflammation and
Accepted 3 October 2013
enhance immunity, whereas greater than 6% of total calories may have adverse effects in
Available online 9 October 2013
rats with subacute peritonitis. Herein, we investigated the effects of parenteral arginine dose on lipid peroxidation (thiobarbituric acid reactive substances, TBARS) and antioxidant
Keywords:
enzyme activities in the plasma and organs.
Peritonitis
Materials and methods: Male Wistar rats with cecal punctureeinduced subacute peritonitis
Arginine
were infused with parenteral nutrition solutions containing 1.61% (CP group), 2.85% (LA
Nitric oxide
group), 4.08% (MA group), and 6.54% (HA group) of total calories as arginine for 7 d. Healthy,
Lipid peroxidation
orally fed rats (NC group) were used as references.
Antioxidant enzymes
Results: Subacute peritonitis significantly elevated the levels of nitrate, nitrite and TBARS in
Nitrotyrosine
the plasma and decreased glutathione peroxidase activity in the kidneys. These changes
Subacute peritonitis
were significantly reversed in the MA and HA groups. The MA and HA groups had signif-
Parenteral nutrition
icantly increased nitrotyrosine levels in the plasma. The LA, MA, and HA groups had significantly increased glutathione peroxidase activity in the plasma, cytochrome P450 levels in the liver, and nitrotyrosine levels in the heart and had significantly decreased TBARS levels in the kidneys compared with the CP group. Conclusions: Our results suggest that parenteral arginine at a dose less than 4% of total calories may attenuate lipid peroxidation and increase antioxidant enzyme activities without leading to nitrosative stress in subacute peritonitis. ª 2014 Elsevier Inc. All rights reserved.
* Corresponding author. Department of Nutritional Science, Fu Jen Catholic University, #510 Zhongzheng Road, Xinzhuang District, New Taipei City, 24205, Taiwan, Republic of China. Tel.: þ886 2 2905 2547; fax: þ886 2 2902 1215. E-mail address: [email protected] (H.-C. Lo). 0022-4804/$ e see front matter ª 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2013.10.002
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1.
Introduction
Peritonitis remains a life-threatening disease, particularly when multiorgan dysfunction develops. It has been hypothesized that the pathogenesis of multiple organ failure in peritonitis is a result of the overproduction of nitric oxide (NO), reactive oxygen species (ROS), and inflammatory cytokines [1e4]. Recent evidence showed that peritonitic patients had increased circulating malondialdehyde and nitrate, that is, the derivatives of lipid peroxidation and NO, and decreased thiols in the ascetic fluid [4]. In peritonitic animals, the levels of antioxidants, such as ascorbic acid (vitamin C) and glutathione (GSH), and the activities of antioxidant enzymes, such as superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx), were significantly decreased in the blood, liver, and lungs [5]. These findings indicated that peritonitis is associated with elevated oxidative stress and weakened antioxidant defense system; thus, a disturbed oxidanteantioxidant homeostasis plays a certain role in the development of multiple organ failure [6]. The peritonitis-induced metabolic changes may result in muscle loss, organ dysfunction, and immune dysregulation [7]. To improve hypercatabolic status, parenteral nutrition has been introduced to patients with peritonitis [8]. However, the prolonged administration of a parenteral solution increases the risk of cholestasis, hepatic steatosis, and liver dysfunction [9] for it may elevate oxidative stress and eliminate antioxidant defenses [10]. Several studies indicated that arginine supplementation may decrease oxidative stress in rats with exhaustive exercise [11], diabetes [12], ulcerative colitis [13], and parenteral feedingeinduced hepatic steatosis [14] and in patients with myocardial ischemic disorders [15], as evidenced by the decreased malondialdehyde levels, the increased GSH levels, and the elevated SOD and catalase activities in the plasma and different organs. These beneficial effects of arginine are closely related to the arginine metabolite NO, a free radical scavenger that may inhibit the activity of pro-oxidant enzymes. Nevertheless, Dioguardi [16] indicated that with greater amounts of arginine introduced, more arginine is destroyed, and impaired NO production eventually occurs in patients with myocardial infarction. This paradox may be related to the production of endogenous inhibitor of nitric oxide synthase, that is, asymmetric dimethylarginine (ADMA) [17]. Using a rat model with subacute peritonitis, that is, nonlethal inflammation, our previous study revealed that the beneficial effects of arginine are dose limited. For example, parenteral arginine may facilitate ureagenesis and proline conversion without resulting in elevated NO levels [18] and may alter leukocytic and splenocytic immunity in an inverted U-shaped dose-dependent manner [19]. These results suggest that arginine replenishment should be carefully evaluated in patients with inflammation, as the arginine-derived metabolite NO may react with superoxide anion to form the toxic compound peroxynitrite [20]. To date, it is still not known whether this dose-dependent effect of parenteral arginine supplementation is associated with changes in oxidanteantioxidant homeostasis. Therefore, in the present study, we investigated the effects of parenteral arginine
135
dosage on the levels of lipid peroxidation, antioxidants, antioxidant enzyme activity, and nitrotyrosine, an indirect marker of peroxynitrite [2], in the plasma and peritonitisaffected target organs, including the heart, lungs, liver, and kidneys of rats with subacute peritonitis.
2.
Methods and materials
2.1.
Experimental protocol
The animal facilities and protocols were reviewed and approved by the Institutional Animal Care and Use Committee in Changhua Christian Hospital, Changhua, Taiwan. Thirty male Wistar rats (National Taiwan University, Taipei, Taiwan) initially weighing approximately 250 g were housed in individual stainless steel cages with free access to water and rat chow (1320 Rat and Mouse Maintenance diet; Altromin GmbH, Germany) in a room maintained at 22 C on a 12:12-h lightedark cycle. After being acclimated for 5 d, six rats maintained on chow diet, water, and nonsurgical procedures were used as normal controls (NC group). The other 24 rats were fasted overnight and anesthetized with intramuscular injection of 100 mg of ketamine and 10 mg of xylazine per kilogram of body weight before surgery (day 0). Subsequently, two surgeries were performed as follows: the placement of catheters in the superior vena cava by way of the external jugular vein for the infusion of parenteral nutrition solutions, and a midline incision in the abdomen with two punctures through both sides of the exposed cecum, as described in our previous study [18]. When making the two cecal punctures, a small amount of fecal material of the size of a rice grain was extruded from each of the puncture sites. After replacing the cecum and fecal material into the abdominal cavity, the abdomen was closed with 3-0 chromic gut sutures for the muscle layer and surgical staples for the skin. After the surgeries, the animals were infused with four different types of parenteral solutions providing equal amounts of calories and 3.97 (standard parenteral solution, CP group), 7.01 (low dose, LA group), 10.58 (medium dose, MA group), or 16.15 (high dose, HA group) grams of arginine per liter of solution for 7 d. The amounts of arginine supplementation accounted for 1.61%, 2.85%, 4.08%, and 6.54% of the total calories in the CP, LA, MA, and HA groups, respectively. During the experimental period, water was provided ad libitum, and the parenteral solutions were infused gradually from 26 mL on day 0 to 62 mL (234 kcal/kg/d) on day 1 through day 6, which is adequate for healthy rats of this size. On day 7, the rats were euthanized under anesthesia via intramuscular injections of 100 mg of ketamine and 10 mg of xylazine per kilogram of body weight. Blood was collected by cardiac puncture, and serum and plasma were obtained for different assays. The liver, lung, heart, and kidneys were dissected and stored at 80 C until further assay.
2.2.
Analytical measurements
To assess the nutritional status and clinical biochemical changes in rats, the serum concentrations of glucose, albumin,
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triglyceride, blood urea nitrogen (BUN), creatinine, glutamic oxaloacetic transaminase (GOT), and glutamic pyruvic transaminase (GPT) were measured by an automatic analyzer (Hitachi 747; Tokyo, Japan). To evaluate the nitrosative status, the plasma concentrations and organ contents of nitrite and nitrate (NOx), which indirectly represent NO, were determined by a commercial colorimetric kit (Cayman Chemical, Ann Arbor, MI). In addition, nitrotyrosine, a specific marker for protein modification by NO-derived oxidants, was measured using an enzyme-linked immunosorbent assay kit (OXIS International, Inc, Oxis Research, Portland, OR). Thiobarbituric acid reactive substances (TBARS), the products of lipid peroxidation as the representative indices of oxidative stress, were determined in the plasma and organs as described by Ohkawa et al. [21]. To determine the levels of sulfhydryl groups, that is, thiol groups, 0.2 mol/L of Tris buffer (pH 8.2) with or without 0.01 mol/L of 5,50 -dithio-bis(2nitrobeznoic acid) was added to the plasma and organ homogenates (3:1 vol/vol). After incubation for 30 min and centrifugation at 3000g for 30 min, the levels of the thiol groups in the supernatants were determined at 412 nm according to the method described by Sedlak and Lindsay [22]. The activities of antioxidant enzymes, that is, SOD, catalase, and GPx, were measured in the plasma and organ homogenates after individual correction of protein concentrations. Protein levels of the plasma and organ homogenates were determined using a commercially available kit (Bicinchoninic Acid Protein Assay; Pierce Chemical Co, Rockford, IL). SOD activity was evaluated according to the inhibition of the pyrogallol autoxidation rate and was expressed in pyrogallol units (U/mg protein) using the method described by Marklund and Marklund [23]. The activity of catalase was determined spectrophotometrically by monitoring the rate of hydrogen peroxide (H2O2) decomposition at 240 nm at 30-s intervals for 3 min using an extinction coefficient of 43.6/(M cm) for H2O2, according to the method described by Aebi [24]. The activity of catalase was expressed as nanomoles of H2O2 decomposition per minute per milligram of protein. The activity of GPx was determined by the method modified from Paglia and Valentine [25] and expressed as nanomoles of reduced nicotinamide adenine dinucleotide phosphate (NADPH) to oxidized nicotinamide adenine dinucleotide phosphate (NADP) per minute per milligram of protein using an extinction coefficient of 6.22 106 for NADPH. The content of cytochrome P450 in the liver microsomes was measured according to the method of Omura and Sato [26]. Microsomes of liver homogenates were extracted and precipitated. The content of cytochrome P450 was determined from the different carbon monoxide spectra of dithionite-reduced samples, assuming a value of 91/(mM cm) for the molar extinction coefficient between 450 and 490 nm, and was expressed as picomoles of per milligram of microsomal protein.
2.3.
Statistical analysis
All treatment groups were compared with a one-way analysis of variance (ANOVA) using the SAS general linear models program. Values are reported as the means standard error of the mean. Group means were considered to be significantly
different at P < 0.05, as determined by the post hoc analysis of protective least-significant difference, in which the ANOVA indicated an overall significant treatment effect at P < 0.05.
3.
Results
3.1.
Parenteral solution infusion and body weights
The amounts of parenteral solution infusion were not significantly different among the four parenterally fed groups with subacute peritonitis during the experimental period (62.4e64.8 kcal/d from day 1 to day 7). After 7 d, the healthy, orally fed rats, that is, the NC group, had a significantly increased body weight (60.71 4.91 g) compared with the peritonitic rats with parenteral nutrition, that is, the CP (0.83 2.68 g), LA (3.00 5.15 g), MA (4.50 3.79 g), and HA (0.17 2.71 g) groups. The final sample size was six rats per group with a 100% survival rate.
3.2.
Clinical biochemical values
Subacute peritonitis was the major factor to alter the clinical biochemical values. Serum concentrations of glucose, albumin, BUN, creatinine, and GPT were significantly decreased in the CP group (i.e., 125.7 4.7 g/100 mL, 32.3 0.7 g/L, 4.64 0.14 mmol/L, 15.9 1.8 mmol/L, and 27.8 4.3 U/L, respectively) compared with the NC group (239.5 3.4 g/ 100 mL, 41.0 1.5 g/L, 8.90 0.50 mmol/L, 33.6 2.7 mmol/L, and 57.3 5.9 U/L, respectively). In contrast, serum concentrations of GOT were significantly increased in the CP group (143.2 9.6 U/L) compared with the NC group (79.0 3.2 U/L). Parenteral arginine did not have a significant impact on these clinical biochemical indices.
3.3.
Relative weights of organs
The relative weights, that is, organ weights divided by body weight on day 7, of the liver, lungs, heart, and kidneys are listed in Tables 2e5, respectively. Peritonitic rats with parenteral nutrition had significantly increased relative weights of the lungs compared with healthy, orally fed rats (P < 0.05). There were no significant differences in the relative weights of the liver, heart, and kidneys among groups.
3.4. Indices of nitrosative and oxidative stress and antioxidant status in the plasma and organs 3.4.1.
Plasma
The levels of NOx, nitrotyrosine, lipid peroxidation products (i.e., TBARS), and antioxidants (i.e., thiol groups), as well as the activities of antioxidant enzymes (i.e., SOD, GPx, and catalase) in the plasma, are shown in Table 1. Parenterally fed rats with subacute peritonitis, that is, the CP, LA, MA, and HA groups, had significantly increased the levels of NOx and TBARS and the CP, LA, and MA groups had significantly decreased the levels of nitrotyrosine and thiol groups in the plasma, compared with healthy, orally fed rats, that is, the NC group (P < 0.05). Peritonitic rats supplemented with medium and high doses of parenteral arginine had significantly
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Table 1 e The levels of NOx, nitrotyrosine, lipid peroxidation, and antioxidants, as well as the activities of antioxidant enzymes in the plasma. Group
NC
NOx, mmol/L Nitrotyrosine, nmol/L TBARS, nmol/mL Thiol group, nmol/mL SOD, U/mg protein GPx, nmol NADPH/min/mg protein Catalase, nmol H2O2/min/mg protein
12.3 8.29 3.64 217.2 310.5 4.01 1.37
CP 0.6 1.15 0.28 8.9 2.6 0.36 0.13
71.8 0.36 7.03 80.3 310.9 0.84 1.17
LA *
4.7 0.13* 0.12* 5.1* 13.4 0.01* 0.05
64.9 1.30 5.87 91.2 435.0 1.70 1.08
MA *
7.3 0.15* 0.53* 8.8* ,y 15.8* *,y 0.25 0.06
49.4 3.81 5.83 65.8 320.9 1.90 1.26
HA *,y
6.0 ,y 0.39* *,y 0.17 5.2* 6.4 ,y 0.13* 0.10
42.6 16.35 4.97 76.6 333.0 3.61 1.10
,y
4.1* ,y 2.89* *,y 0.55 4.8* 6.2 y 0.36 0.02
Values are represented as the means standard error of the mean; n ¼ 6 for each group. * Values are significantly different from the NC group at P < 0.05 by a one-way ANOVA. y Values are significantly different from the CP group at P < 0.05 by a one-way ANOVA.
differences in the contents of TBARS and thiol groups and the activities of SOD, GPx, or catalase in the lungs among groups.
reversed changes in plasma NOx, nitrotyrosine, and TBARS. Parenterally fed rats with subacute peritonitis had significantly decreased plasma GPx activities, and the low, medium, and high doses of parenteral arginine supplementation significantly attenuated these decreases. In addition, a low dose of arginine supplementation significantly increased plasma SOD activities in peritonitic rats. Neither peritonitis nor arginine supplementation had a significant impact on plasma catalase activity.
3.4.2.
3.4.4.
Liver
The NOx and nitrotyrosine contents, as well as the catalase activity, were significantly increased, whereas the thiol group contents and the GPx activity were significantly decreased in the liver in the CP, LA, MA, and HA groups compared with the NC group (Table 2). The LA, MA, and HA groups had 2-, 2-, and 4-fold increases in cytochrome P450 content in the liver compared with the NC and CP groups. There were no significant differences in the contents of TBARS in the liver among groups.
3.4.5. 3.4.3.
Heart
The concentrations of nitrotyrosine and TBARS and the activities of SOD, GPx, and catalase in the heart were significantly decreased in the CP group compared with the NC group (Table 4). The LA group had significantly increased the contents of NOx and TBARS, whereas the MA group had significantly increased NOx contents in the heart compared with the CP group. In addition, the peritonitis-induced decreases in the contents of nitrotyrosine were significantly elevated by parenteral arginine supplementation, as shown by the 4-, 4-, and 2-fold increases in the LA, MA, and HA groups, respectively, compared with the CP group. There were no significant differences in the contents of thiol groups in the heart among groups. In addition, parenteral arginine supplementation had no significant impact on the activities of SOD, GPx, or catalase in the hearts of rats with subacute peritonitis.
Kidneys
The contents of NOx and thiol groups were significantly increased, and the activity of GPx was significantly decreased in the kidneys of the CP group compared with the NC group (Table 5). The contents of nitrotyrosine were significantly increased in the LA and MA groups compared with the NC and CP groups. Parenteral arginine significantly decreased the
Lungs
The concentrations of NOx and nitrotyrosine in the lungs were significantly increased in the CP group compared with the NC group (Table 3). Peritonitic animals supplemented with a medium dose of parenteral arginine had a significantly decreased NOx content in the lungs. However, there were no significant
Table 2 e The relative weights, levels of NOx, nitrotyrosine, lipid peroxidation, and antioxidants, and activities of antioxidant enzymes in the liver. Group Relative weight, g/kg body weight NOx, mmol/g tissue Nitrotyrosine, pmol/mg protein TBARS, nmol/g tissue Thiol group, nmol/g tissue SOD, U/mg protein GPx, nmol NADPH/min/mg protein Catalase, nmol H2O2/min/mg protein Cytochrome P450, pmol/mg protein
NC 51.29 255 3.28 514.3 20.90 141.9 546.8 43.16 8.31
CP 2.90 16 0.33 30.0 0.71 8.5 46.2 10.8 2.02
46.91 366 5.28 492.1 17.29 186.4 297.3 309.1 9.33
LA 2.18 11* 0.22* 36.4 0.76* 11.7 26.3* 31.8* 1.17
47.40 383 5.50 524.8 17.44 253.8 336.6 242.4 17.07
Values are represented as the means standard error of the mean; n ¼ 6 for each group. * Values are significantly different from the NC group at P < 0.05 by a one-way ANOVA. y Values are significantly different from the CP group at P < 0.05 by a one-way ANOVA.
2.07 24* 0.61* 35.4 0.48* 20.8* 19.3* 23.4* 2.58*,y
MA 42.95 392 6.07 483.2 17.11 222.9 321.5 278.6 17.97
2.73 12* 0.49* 46.0 0.73* 27.0* 11.7* 21.2* 2.30*,y
HA 46.82 351 6.54 502.8 18.11 196.2 281.3 402.4 41.10
0.74 11* 0.70* 45.3 0.34* 18.4 9.5* 53.8* 3.29*,y
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Table 3 e The relative weights, levels of NOx, nitrotyrosine, lipid peroxidation, and antioxidants, and activities of antioxidant enzymes in the lung. Group
NC
Relative weights, g/kg body weight NOx, mmol/g tissue Nitrotyrosine, pmol/mg protein TBARS, nmol/g tissue Thiol group, nmol/g tissue SOD, U/mg protein GPx, nmol NADPH/min/mg protein Catalase, nmol H2O2/min/mg protein
6.37 299 17.5 319.5 8.20 74.26 538.9 1075
0.26 44 2.0 32.9 0.81 10.31 45.8 54
CP 8.69 491 30.2 394.9 7.32 92.06 596.7 1067
LA 0.32 35* 2.7* 21.7 0.40 7.02 46.4 30
*
9.14 419 28.8 389.2 7.53 84.78 551.5 981
MA *
0.35 47* 3.2* 27.8 0.33 5.81 53.2 62
8.18 366 33.8 364.9 7.78 90.72 590.9 1010
0.38 30y 2.9* 24.9 0.69 3.75 51.3 56
HA *
8.60 435 34.2 347.2 8.19 99.03 550.0 1021
0.18* 20* 3.6* 12.6 0.16 5.80 38.5 33
Values are represented as the means standard error of the mean; n ¼ 6 for each group. * Values are significantly different from the NC group at P < 0.05 by a one-way ANOVA. y Values are significantly different from the CP groups at P < 0.05 by a one-way ANOVA.
contents of TBARS and thiol groups in the kidneys in a dosedependent manner. In addition, the peritonitis-induced decreases in the GPx activity of the kidneys were significantly increased in rats supplemented with medium and high doses of parenteral arginine. There were no significant differences in the activities of SOD and catalase in the kidneys among groups.
4.
Discussion
It has been demonstrated that activated leukocytes and neutrophils may produce excessive ROS and NO to ignite inflammatory response in trauma and sepsis, which, in turn, lead to organelle dysfunction and multiple organ failure [6]. Conditions of arginine deficiency worsen protein metabolism, microcirculation, immune function, and antioxidant status in sepsis [27]. However, arguments have been raised against arginine supplementation concerning the toxicity of excess NO and hemodynamic instability. Previous studies have shown that parenteral arginine at a dose of 6.54% of the total calories may not elevate circulating NOx levels [18] and that 2%e4% of total calories may enhance the immunities of peripheral blood cells and splenocytes in rats with subacute peritonitis [19]. In this study, we showed that parenteral arginine may attenuate lipid peroxidation and elevate GPx activity, although potentially increasing the NOx and
nitrotyrosine contents in the plasma and in certain organs in rats with subacute peritonitis. In the present study, subacute peritonitic rats with parenteral feeding had significantly lower body weight gains; indices of nutrition status, such as serum glucose, albumin, and triglyceride; and serum BUN and creatinine levels compared with healthy rats with oral feeding. Our previous study showed that rats with subacute peritonitis have significantly decreased serum insulin-like growth factor (IGF)-I, a commonly used anabolic hormone to assess nutrition status that has an insulin-like action and a short half-life (10e20 min) [18]. These results implied that rats with subacute peritonitis may be in some degree of undernutrition. When supplemented with a low dose of parenteral arginine, the peritonitis-induced decrease in serum IGF-I was attenuated [18], but body weight, serum albumin, and serum insulin were not significantly affected. The relative lung weights (Table 3), serum GOT, and cytochrome P450 content in the liver (Table 2) were significantly increased, and the serum GPT was significantly decreased by parenteral arginine. These changes suggested that although parenteral arginine supplementation may not improve whole body catabolism, it still has certain effects on the lung, heart, liver, and kidneys in rats with subacute peritonitis. Evidence has shown that patients with septic shock and secondary organ dysfunction showed significantly decreased plasma antioxidant status, increased plasma lipid peroxidation, and elevated urinary nitrite excretion [6,28]. In subacute
Table 4 e The relative weights, levels of NOx, nitrotyrosine, lipid peroxidation, and antioxidants, and activities of antioxidant enzymes in the heart. Group Relative weights, g/kg body weight NOx, mmol/g tissue Nitrotyrosine, pmol/mg protein TBARS, nmol/g tissue Thiol group, nmol/g tissue SOD, U/mg protein GPx, nmol NADPH/min/mg protein Catalase, nmol H2O2/min/mg protein
NC 3.60 243 50.46 597.2 13.04 191.0 539.4 1011
CP 0.11 22 4.83 48.2 0.24 18.1 25.6 40
3.96 207 26.74 490.1 13.15 142.1 376.8 689
LA 0.26 18 7.56* 28.0* 0.42 3.3* 16.7* 24*
4.18 423 110.35 585.1 11.62 128.9 399.9 722
Values are represented as the means standard error of the mean; n ¼ 6 for each group. * Values are significantly different from the NC group at P < 0.05 by a one-way ANOVA. y Values are significantly different from the CP group at P < 0.05 by a one-way ANOVA.
0.14 ,y 13* ,y 15.19* y 24.8 0.67 6.6* 27.5* 17*
MA 4.32 292 103.88 547.9 12.21 127.0 385.4 756
0.35 22y ,y 16.94* 24.4 0.76 4.8* 20.6* 33*
HA 4.01 207 56.92 507.6 12.14 147.9 394.7 780
0.12 11 ,y 8.39* * 17.3 0.52 6.1* 13.0* 32*
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Table 5 e The relative weights, levels of NOx, nitrotyrosine, lipid peroxidation, and antioxidants, and activities of antioxidant enzymes in the kidneys. Group Relative weight, g/kg body weight NOx, mmol/g tissue Nitrotyrosine, pmol/mg protein TBARS, nmol/g tissue Thiol group, nmol/g tissue SOD, U/mg protein GPx, nmol NADPH/min/mg protein Catalase, nmol H2O2/min/mg protein
NC 10.97 139 87.6 419.7 13.01 270.6 1112 566.8
CP 0.54 5 6.0 9.0 1.19 9.6 117 44.1
10.34 250 148.6 383.2 15.08 265.7 902 652.7
LA 0.16 27* 18.7 8.7 0.46* 8.6 42* 28.5
10.98 138 347.7 290.5 13.12 294.1 1022 660.8
0.37 18 ,y 39.5* *,y 22.8 0.34y 8.4 63 24.3
MA 10.44 251 465.2 265.2 12.77 281.4 1157 591.2
0.59 35* ,y 44.9* *,y 36.2 0.29y 10.8 45y 57.9
HA 11.06 264 110.8 238.1 11.94 290.8 1104 638.0
0.13 54* 9.9 ,y 30.8* y 0.51 10.3 51y 29.4
Values are represented as the means standard error of the mean; n ¼ 6 for each group. * Values are significantly different from the NC group at P < 0.05 by a one-way ANOVA. y Values are significantly different from the CP groups at P < 0.05 by a one-way ANOVA.
peritonitic rats with parenteral nutrition, the levels of NOx and TBARS in the plasma were significantly increased, whereas nitrotyrosine and thiol group levels in the plasma, the physiological free radical scavengers, and GPx activities were significantly decreased (Table 1). These changes revealed that these rats had a systemic imbalance in oxidanteantioxidant homeostasis. A similar imbalance was found in the organs of these rats, such as the significant increases in NOx levels in the liver, lungs, and kidneys and the significant decreases in thiol group levels in the liver, GPx activity in the liver, heart, and kidneys, and SOD and catalase activities in the heart. These results indicated that subacute peritonitic rats with parenteral nutrition were under conditions of systemic and local oxidative stress and antioxidant defense defects. The beneficial effects of arginine on oxidanteantioxidant homeostasis have been observed in inflammation and sepsis [13e15]. It has been proposed that the “arginine paradox” may impair NO production because of the endogenous inhibitor of nitric oxide synthase, that is, ADMA [16]. In the present study, parenteral arginine supplementation at 4%e6%, but not at 2%, of the total calories significantly alleviated the increases in the levels of NOx and TBARS in the plasma in the peritonitic rats. These results suggest that parenteral arginine at 4%e6% of the total calories may represent a condition of the arginine paradox; however, we did not measure the levels of ADMA in the present study to prove it. It is also possible that argininederived NO reacts with superoxide to decrease the production of lipid peroxidation, as shown in exhaustively exercised rats fed with a diet supplemented with 2% arginine [29]. Arginine supplementation may reduce the exercise-induced oxidanteantioxidant imbalance and this modulation is likely mediated through the arginineeNO pathways [30]. However, the increased accumulation of nitrotyrosine, a product generated by NO and superoxide (O 2 ), in the kidneys was found to be mainly because of the oxidation of NO by ROS in rats with GSH depletion-induced hypertension [31]. In this study, subacute peritonitic rats had significantly decreased plasma thiol levels and GPx activity, and parenteral arginine significantly elevated the plasma nitrotyrosine levels and GPx activities, whereas decreasing the levels of NOx and TBARS in the plasma. These results support the concept that the arginine-derived NO may react with superoxides to decrease the production of lipid peroxidation in subacute peritonitic rats with parenteral arginine supplementation. However, the
arginine-induced increase in plasma nitrotyrosine may still be able to cause adverse effects. In septic rats, Mu¨ller et al. [32] found that plasma NOx concentrations were negatively correlated with the total cytochrome P450 content in the liver microsomes. In this study, peritonitic rats supplemented with parenteral arginine at 2.85%, 4.08%, and 6.54% of total calories had 2-, 2-, and 4-fold increases in the total cytochrome P450 content in the liver and 0.9-, 0.7-, and 0.6-fold increases in plasma NOx levels compared with those with parenteral arginine as 1.61% of total calories. We confirmed that there is a negative relationship between the plasma NOx and total cytochrome P450 content in the liver of subacute peritonitic rats with parenteral arginine supplementation. In addition, we found that greater than 6% of total calories as parenteral arginine may result in a greater increase in the oxidation of organic substances in the liver. Oxidanteantioxidant homeostasis in the body is sensitively controlled by two antioxidant defense systems, that is, nonenzymatic antioxidants, including vitamins, minerals, cofactors, and thiols, and enzymatic antioxidants, including SOD, catalase, GPx, and GSH reductase. In conditions of trauma, burn, or sepsis, oxygen-derived free radicals are excessively produced, activating SOD to excessively catalyze the conversion of superoxide into H2O2. Then, H2O2 is further detoxified by catalase and GPx [27,33]. Warner et al. [34] found that septic patients had significantly elevated total plasma SOD activity compared with healthy controls, whereas septic survivors had significantly lower total plasma SOD and catalase activities than septic nonsurvivors. In addition, the elevated activities of serum SOD and GPx and the increased extent of lipid peroxidation were observed in septic neonates [35]. In the present study, subacute peritonitic rats did not show significant changes in the plasma activities of SOD and catalase but instead had significantly decreased plasma GPx activity and increased plasma TBARS levels. These inconsistent changes in antioxidant enzymes might be because of differing severities and phases of sepsis, as suggested by Andrades et al. [36]. These changes in antioxidant enzymes seem to differ between nonlethal and lethal sepsis. Our findings also showed that subacute peritonitic rats supplemented with parenteral arginine may have elevated plasma SOD and GPx activities and decreased plasma lipid peroxidation, which suggest that parenteral arginine may partially alleviate systemic oxidative stress.
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A recent study reported that oxidanteantioxidant homeostasis may be varied in the circulation and organs, depending on the physiological functions and the degree of oxidative damage [36]. For example, catalase activity was decreased in the livers of septic rats with cecal ligation and punctures [37]. In addition, TBARS were positively correlated with lung and renal damage, whereas oxidative damage was only correlated with an increase in the ratio of SOD to catalase in the lungs, but not in kidneys, of the lethal septic rats [38]. However, the increases in the ratio of SOD to catalase in the heart, liver, lung, and kidneys of the nonlethal septic rats were much lower than those of the lethal septic rats [36]. In subacute peritonitic rats, a nonlethal septic rat model, we found that GPx activity was decreased, catalase activity and the cytochrome P450 content were increased, and the content of TBARS was not changed in the liver. These results suggest that an oxidanteantioxidant balance in the liver may be achieved. In the heart, the activities of SOD, GPx, and catalase were significantly decreased. Accompanied with the increased serum GOT levels, we speculated that these subacute peritonitic rats might exhibit a certain degree of heart damage; however, we did not measure heart function to confirm it. Herein, we showed different changes in the activities of antioxidant enzymes, revealing that oxidanteantioxidant homeostasis may vary across different organs. In addition, parenteral arginine supplementation at a dose greater than 4% of the total calories can increase the GPx activity in the kidneys. Whether this effect is associated with the physiological function of the kidneys in arginine metabolism needs further investigation. The present study has several limitations. First, there were no sham-operated and parenteral-fed rats as controls to distinguish between the individual effects of parenteral feeding and subacute peritonitis on the oxidative stress and antioxidant defense system. Therefore, the results of the CP group represented the combined effects of parenteral feeding and subacute peritonitis. Second, we only obtained samples on day 7 to investigate the long-term effects of parenteral arginine supplementation. Thus, we might have overlooked more immediate effects of parenteral arginine. Third, we did not measure the protein or messenger RNA levels of these antioxidant enzymes. It might be possible to demonstrate the effects of parenteral arginine on the molecular levels of these enzymes. Finally, more assays should be performed; for example, blood gas assays for lung function and cardiac indices for heart performance would facilitate the investigation of whether parenteral arginine supplementation improves organ function via the changes in the oxidative stress and antioxidant defense systems.
5.
Conclusions
The results of this study revealed that subacute peritonitis significantly increased systemic and local oxidative and nitrosative stresses and diminished the antioxidant defense system. In addition, parenteral arginine at a dose of 4%e6% of the total calories may attenuate lipid peroxidation and elevate antioxidant enzyme activity and nitrosative stress.
Therefore, parenteral arginine supplementation may maintain oxidanteantioxidant homeostasis in a dose- and organdependent manner. To sum up, parenteral arginine at a dose less than 4% of total calories may attenuate lipid peroxidation and increase antioxidant enzyme activities through L-arginineeNO pathways without leading to nitrosative stress in subacute peritonitis.
Acknowledgment This work was supported by the National Science Council of the Republic of China under the grant number NSC-95-2320-B309-001 and by Changhua Christian Hospital under the grant number CCH-92106.
references
[1] Kim YM, Hong SJ, Billiar TR, Simmons RL. Counterprotective effect of erythrocytes in experimental bacterial peritonitis is due to scavenging of nitric oxide and reactive oxygen intermediates. Infect Immun 1996;64:3074. [2] Xie YW, Wolin MS. Role of nitric oxide and its interaction with superoxide in the suppression of cardiac muscle mitochondrial respiration. Involvement in response to hypoxia/reoxygenation. Circulation 1996;94:2580. [3] Cuzzocrea S, Zingarelli B, Costantino G, Sottile A, Teti D, Caputi AP. Protective effect of poly(ADP-ribose) synthetase inhibition on multiple organ failure after zymosan-induced peritonitis in the rat. Crit Care Med 1999;27:1517. [4] Natarajan SK, Mukhopadhya A, Ramachandran A, Amalanathan S, Kurian G, Balasubramanian KA. Spontaneous bacterial peritonitis results in oxidative and nitrosative stress in ascitic fluid. J Gastroenterol Hepatol 2007;22:177. [5] Demling R, Nayak U, Ikegami K, LaLonde C. Comparison between lung and liver lipid peroxidation and mortality after zymosan peritonitis in the rat. Shock 1994;2:222. [6] Vı´ctor VM, Espulgues JV, Herna´ndez-Mijares A, Rocha M. Oxidative stress and mitochondrial dysfunction in sepsis: a potential therapy with mitochondria-targeted antioxidants. Infect Disord Drug Targets 2009;9:376. [7] Michie HR. Metabolism of sepsis and multiple organ failure. World J Surg 1996;20:460. [8] Ikeda K, Kimura Y, Iwaya T, et al. Perioperative nutrition for gastrointestinal surgery. Nippon Geka Gakkai Zasshi 2004; 105:218. [9] Weinberger B, Watorek K, Strauss R, Witz G, Hiatt M, Hegyi T. Association of lipid peroxidation with hepatocellular injury in preterm infants. Crit Care 2002;6:521. [10] Cai W, Wu J, Hong L, Xu Y, Tang Q, Shi C. Oxidative injury and hepatocyte apoptosis in total parenteral nutrition-associated liver dysfunction. J Pediatr Surg 2006;41: 1663. [11] Huang CC, Lin TJ, Lu YF, Chen CC, Huang CY, Lin WT. Protective effects of L-arginine supplementation against exhaustive exercise-induced oxidative stress in young rat tissues. Chin J Physiol 2009;52:306. [12] Coronel I, Arellano-Mendoza MG, del Valle-Mondragon L, et al. L-arginine and antioxidant diet supplementation partially restores nitric oxide-dependent regulation of phenylephrine renal vasoconstriction in diabetics rats. J Ren Nutr 2010;20:158.
j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 7 ( 2 0 1 4 ) 1 3 4 e1 4 1
[13] Harisa GE, Abo-Salem OM, El-Sayed el-SM, Taha EI, ElHalawany N. L-arginine augments the antioxidant effect of garlic against acetic acid-induced ulcerative colitis in rats. Pak J Pharm Sci 2009;22:373. [14] Ilan E, Tirosh O, Madar Z. Triacylglycerol-mediated oxidative stress inhibits nitric oxide production in rat isolated hepatocytes. J Nutr 2005;135:2090. [15] Tripathi P, Misra MK. Therapeutic role of L-arginine on free radical scavenging system in ischemic heart diseases. Indian J Biochem Biophys 2009;46:498. [16] Dioguardi FS. To give or not to give? Lessons from the arginine paradox. J Nutrigenet Nutrigenomics 2011;4:90. [17] Bo¨ger RH. Asymmetric dimethylarginine, an endogenous inhibitor of nitric oxide synthase, explains the “l-arginine paradox” and acts as a novel cardiovascular risk factor. J Nutr 2004;134:2842S. [18] Lo HC, Wu SC, Li ML, Lee CH. Does pharmacological dose of parenteral arginine have beneficial effect in rats with subacute peritonitis? Pediatr Surg Int 2010;26:625. [19] Chen YH, Lee CH, Hsu LS, Hsiao CC, Lo HC. Appropriate dose of parenteral arginine enhances immunity of peripheral blood cells and splenocytes in rats with subacute peritonitis. JPEN J Parenter Enteral Nutr 2012;36:741. [20] Pacher P, Beckman JS, Liaudet L. Nitric oxide and peroxynitrite in health and disease. Physiol Rev 2007;87:315. [21] Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979;95:351. [22] Sedlak J, Lindsay RH. Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman’s reagent. Anal Biochem 1968;25:192. [23] Marklund S, Marklund G. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem 1974;47:469. [24] Aebi HE. Catalase. In: Bergmeyer HU, editor. Methods of enzymatic analysisvol. 3. Weinheim: VCH Verlag; 1987. p. 273e8. [25] Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 1979;70:158. [26] Omura T, Sato R. The carbon monoxide-binding pigment of liver microsomes. J Biol Chem 1964;239:2379.
141
[27] Luiking YC, Deutz NE. Exogenous arginine in sepsis. Crit Care Med 2007;35(9 Suppl):S557. [28] Goode HF, Cowley HC, Walker BE, Howdle PD, Webster NR. Decreased antioxidant status and increased lipid peroxidation in patients with septic shock and secondary organ dysfunction. Crit Care Med 1995;23:646. [29] Lin WT, Yang SC, Tsai SC, Huang CC, Lee NY. L-arginine attenuates xanthine oxidase and myeloperoxidase activities in hearts of rats during exhaustive exercise. Br J Nutr 2006;95:67. [30] Shan L, Wang B, Gao G, Cao W, Zhang Y. L-arginine supplementation improves anti-oxidant defenses through L-arginine/nitric oxide pathways in exercise rats. J Appl Physiol; 2013 [Epub ahead of print]. [31] Zhou XJ, Vaziri ND, Wang XQ, Silva FG, Laszik Z. Nitric oxide synthase expression in hypertension induced by inhibition of glutathione synthase. J Pharmacol Exp Ther 2002;300:762. [32] Mu¨ller CM, Scierka A, Stiller RL, et al. Nitric oxide mediates hepatic cytochrome P450 dysfunction induced by endotoxin. Anesthesiology 1996;84:1435. [33] Guemouri L, Artur Y, Herbeth B, Jeandel C, Cuny G, Siest G. Biological variability of superoxide dismutase, glutathione peroxidase, and catalase in blood. Clin Chem 1991;37:1932. [34] Warner A, Bencosme A, Healy D, Verme C. Prognostic role of antioxidant enzymes in sepsis: preliminary assessment. Clin Chem 1995;41(6 Pt 1):867. [35] Barbas C, Castro M, Bonet B, Viana M, Herrera E. Simultaneous determination of vitamins A and E in rat tissues by high-performance liquid chromatography. J Chromatogr A 1997;778:415. [36] Andrades M, Ritter C, Moreira JC, Dal-Pizzol F. Oxidative parameters differences during non-lethal and lethal sepsis development. J Surg Res 2005;125:68. [37] Llesuy S, Evelson P, Gonza´lez-Flecha B, et al. Oxidative stress in muscle and liver of rats with septic syndrome. Free Radic Biol Med 1994;16:445. [38] Andrades M, Ritter C, de Oliveira MR, Streck EL, Fonseca Moreira JC, Dal-Pizzol F. Antioxidant treatment reverses organ failure in rat model of sepsis: role of antioxidant enzymes imbalance, neutrophil infiltration, and oxidative stress. J Surg Res 2011;167:e307.
