- Email: [email protected]
The impact of arginine on bacterial translocation in an intestinal obstruction model in rats
ARTICLE IN PRESS Clinical Nutrition (2007) 26, 335–340
Available at www.sciencedirect.com
http://intl.elsevierhealth.com/journals/clnu
ORIGINAL ARTICLE
The impact of arginine on bacterial translocation in an intestinal obstruction model in rats Iara Eliza Pacı´fico Quirinoa, Maria Isabel Toulson Davisson Correiab,, Valbert Nascimento Cardosoa a
School of Pharmacy, Federal University of Minas Gerais, Brazil School of Medicine, Federal University of Minas Gerais, Av. Carandaı´ 246 apt. 902, Belo Horizonte, MG 30130-060, Brazil
b
Received 10 July 2006; accepted 19 December 2006
KEYWORDS Arginine; Bacterial translocation; Morbidity; Mortality
Summary Background & aims: Arginine has been shown to have multiple beneficial metabolic and immunologic effects in stress situations. Supplementation of arginine has been shown to promote wound healing and intestinal mucosal recovery after trauma, ischemia or intestinal resection. Bacterial translocation has also been evaluated although with conflicting results and using different assessing techniques. Therefore, the aim of this study was to evaluate the effects of arginine on bacterial translocation in an intestinal obstruction model in rats using Escherichia coli labeled with 99mTechnetium. Methods: Male Wistar rats (250–350 g) were randomized to receive conventional chow, diet supplemented with pure arginine or diet supplemented with an immunonutrition enteral formula, enriched with arginine, omega-3 fatty acid and RNA. After 7 days, the animals were anesthetized. Terminal ileum was isolated and a ligature was placed around it. E. coli labeled with 99mTechnetium (99mTc-E. coli) was inoculated into the intestinal lumen (terminal ileum). After 24 h, the animals were sacrificed. Blood, mesenteric lymph nodes (MLN), liver, spleen and lungs were removed for radioactivity determination. Results: Arginine supplementation (300 mg/day, 600 mg/day or present in the enteral formula) reduced the level of bacterial translocation when compared with the control group (po0.05). This was shown by significantly decrease uptake of 99mTc-E. coli in blood, MLN, liver, spleen and lungs of the animals in the experimental groups (po0.05).
Corresponding author. Tel.: +55 31 91688239; fax: +55 31 32411367.
E-mail addresses: [email protected] (I.E.P. Quirino), [email protected] (M.I.T.D. Correia), [email protected] (V.N. Cardoso). 0261-5614/$ - see front matter & 2007 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved. doi:10.1016/j.clnu.2006.12.007
ARTICLE IN PRESS 336
I.E.P. Quirino et al. Conclusions: These results have shown that arginine was able to decrease bacteria translocation despite intestinal obstruction. There are several mechanisms which might explain the role of arginine and these will be the subject of future studies. & 2007 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.
Background Nutrition has usually been seen as the supply of energy and nitrogen to maintain body mass. Recent studies have, however, revealed that nutrition also plays an important role in modulating the immune and inflammatory responses. The clinical relevance of this modulation has led to the investigation of diets containing specific substrates the socalled immunonutrients, such as glutamine, arginine, nucleotides and omega-3 fatty acids.1 These formulas have been related to modulation of gut function, reduction of infectious complications as well as hospital stay and ventilator days in critically ill patients.2,3 However, because these diets contain more than one immunonutrient, it is difficult to determine which of these elements plays the key role in modulating such phenomena. Thus, attempts to assess the individual role of each of them in different settings are crucial. Arginine, a semiessential amino acid, has been shown to have multiple beneficial metabolic and immunologic effects, especially in stress situations. It seems to promote nitrogen retention and wound healing in the form of increased wound tensile strength and reparative collagen deposition.4 Arginine is important for physiologic functions of immune cells. Ochoa et al.5 observed that T lymphocytes grown in arginine deficient media exhibited decreased membrane expression of T-cell receptors and deficient phenotypic differentiation toward the formation of memory T-cells. Arginine is also necessary for the normal function of myeloid cells such as macrophages and dendritic cells. Myeloid cell activation, such as in critical conditions, induces the expression of membrane arginine transport mechanism and increases arginine uptake. The products of arginine’s metabolism are polyamines (via arginase) and nitric oxide (via nitric oxide synthase). Polyamines such as putrescine, spermine and spermidine act in membrane transport, cell growth, cell proliferation and cell differentiation. Nitric oxide is a vasodilator and an antimicrobial agent effective against intracellular pathogens, extracellular parasites and bacteria. The disease process will determine the metabolic pathways and the ultimate metabolic fate of arginine in the immune system.6 Some studies have shown that arginine decreases bacteria translocation in radiation enteritis and peritonitis animal models whose diets were supplemented with it.4,7,8 However, Samel et al.9 observed that when arginine was given intravenously, bacterial translocation was increased. The exact role exerted by arginine in the process of bacterial translocation is still unclear. This fact may be due to the many mechanisms enrolled in bacterial translocation associated to different critical situations such as hemorrhagic shock, burns, malnutrition, intestinal obstruction, cirrhosis, jaundice, acute pancreatitis, prolonged use of antibiotics,
total parenteral nutrition and magnitude of the operation. Ischemia, increase of intestinal permeability and overgrowth of enteric bacteria are observed in these conditions and result in the spread of bacteria from the gut to other organs.4,10–12 Translocation of bacteria and endotoxins across a leaky mucosa may be responsible for the release of systemic mediators and activation of immunologic cells that contribute to the development of systemic inflammation and multiple organ failure. This is a major cause of death in critically ill patients.1,6,12 In the present study, the role of arginine on bacterial translocation from the intestinal lumen to sterile organs such as liver, spleen, lungs and mesenteric lymph nodes in an intestinal obstruction model assessed by Escherichia coli labeled with 99mTechnetium was investigated.
Methods Animals Forty male Wistar rats weighting between 250 and 350 g were included in this study. The animals were randomized to five groups of eight animals each one: (1) ‘‘sham’’ operated (no obstruction; inoculation of 12.5 MBq of 99mTechnetiumEscherichia coli (99mTc-E. coli)) fed a conventional chow diet (LABINAs, Purina, Brazil; 23% protein); (2) IO group fed a conventional chow diet, submitted to intestinal obstruction and inoculation of 12.5 MBq of 99mTc-E. coli; (3) Arginine 300 mg group fed conventional chow supplemented with arginine 300 mg/day (L-arginine, Ajinomoto do Brazil, Sa ˜o Paulo, Brazil), submitted to intestinal obstruction and inoculation of 12.5 MBq 99mTc-E. coli; (4) Arginine 600 mg group fed conventional chow supplemented with Arginine 600 mg/day, submitted to intestinal obstruction and inoculation of 12.5 MBq of 99mTc-E. coli; (5) Impacts group fed conventional chow supplemented with enteral formula Impacts (Novartis, Rio de Janeiro, Brazil; 22% protein) amounting 600 mg/day of arginine, submitted to intestinal obstruction and inoculation of 99mTc-E. coli. The animals were fed these diets for 7 days previous to intestinal injury and were monitored daily in terms of ingestion and body weight. The supplemented diets were offered first and only when they were finished were the animals allowed conventional chow ad libitum. Water was given ad libitum. The research protocol was approved by the Ethical Committee of Animal Research of Federal University of Minas Gerais.
Preparation of the supplemented diet Conventional rat chow was ground to powder (89%) and added to arginine powder (6%), colorless gelatin powder (4%), corn
ARTICLE IN PRESS Arginine and bacterial translocation starch (1%) and water. Chow powder and arginine powder were homogenized with dissolved gelatin (warm water was used in this process) and extra water was added to guarantee the homogenization. The homogenized diet mass was then divided in small cylinders (pellets). These cylinders were dried in an oven at 45 1C for 8 h. For the Impacts supplemented diet, conventional chow powder (38.1%) was added to the enteral formula (61.9%) and water. The percentage of carbohydrates was derived by the following equation: carbohydrate ¼ total solids (protein+fat+ashes). It was considered that 100 g of protein was equal to 16 g of nitrogen.
Radiolabeling of E. coli A sample of E. coli ATCC-10536 culture grown overnight in tripticasein agar was transferred to 10 mL of saline solution. Bacterial concentration of 108 CFU/mL was adjusted spectophotometrically in 31% of transmittance in 580 nm. An aliquot of bacterial solution (2 mL) was incubated in tubes with 1 mL of stannous chloride solution (580 mM, pH 7.0) at 37 1C for 10 min. After incubation, 1.0–1.5 mCi of 99mTc were added and the preparation was kept at 37 1C for 10 min. The tubes were then centrifuged at 3000 g for 25 min and 100 mL of the supernatant and 100 mL of re-suspended precipitate in saline were used to measure radioactivity. This procedure was repeated three times. The 99mTc percent incorporated into bacterial cells was determined using the following equation13: % labeled bacteria cpm of precipitate 100, ¼ cmp of precipitate þ cpm of supernatant where cpm is the counts per minute.
Surgical procedure Seven days after the beginning of the study, the animals were anesthetized intraperioneally with tiazine (8.7 mg/kg) and ketamine (25.2 mg/kg) solutions. The abdomen was opened through a midline incision and the terminal ileum was isolated and ligated.14 The solution with labeled 99mTcE. coli was inoculated into the distal ileum. The abdominal wound was closed in two layers.
Bacterial translocation analysis Twenty-four hours later, the animals were once again anesthetized using the same technique. Blood (7 mL), Table 1
337 mesenteric lymph nodes (MLN), liver, spleen and lungs were removed, weighed, washed and placed in tubes for determination of radioactivity.15
Statistical analysis Bacterial translocation: statistical analysis among the groups was performed using the Kruskall–Wallis analysis of variance and a post hoc analysis using the Dunn’s test. Data are expressed as median. Differences were considered statistically significant when p values were less than 0.05. Weight, caloric, protein, nitrogen and arginine intake were compared using the ANOVA test and a post hoc analysis using the Duncan’s test. Data were expressed as mean7 standard deviation (SD). Differences were considered statistically significant when p values were less than 0.05. All analysis were performed using the program ‘‘BioEstat Version 3.0 (Mamiraua ´ Civil Society/MCT – CNPq)’’.
Results Composition of standard chow and supplemented chows The composition of standard and supplemented chows is shown in Table 1. There was an increase in total calories in the Impact formula (355.6 kcal/100 g) compared to the conventional chow (275 kcal/100 g) and the arginine supplemented chow (264.3 kcal/100 g). There was an increase of protein, nitrogen and arginine in the chow supplemented with arginine and an increase of arginine, fat and total calories in the chow supplemented with the enteral formula. Carbohydrate content was slight reduced in all supplemented diets when compared to the standard chow.
Caloric ingestion, protein ingestion and weight gain The Impacts group had higher caloric ingestion (Fig. 1) than the other groups (po0.05). Protein and nitrogen intake (Figs. 2 and 3) were higher in Arginine 300 mg and Arginine 600 mg groups (po0.05). Arginine 600 mg group showed higher arginine intake, followed by Impacts and Arginine 300 mg groups (Fig. 4). Arginine represented 1.8% of the total caloric value in the Arginine 300 mg group, 3.3% in the 600 mg and 3.1% in the Impact group. Despite these
Composition of standard chow and supplemented diets (percents).
Nutrients/calories
Standard chow
Supplemented chow with arginine
Supplemented chow with enteral formula
Carbohydrates Fat Protein Nitrogen Arginine Water, ashes and fiber
63.0 4.0 23.0 3.7 1.5 10.0
56.1 3.5 28.1 4.5 6.6 13.3
56.9 8.2 22.9 3.7 3.7 12.0
ARTICLE IN PRESS 338
I.E.P. Quirino et al. 1.20
85.0 ab
80.0
a
a 1.00 Arginine intake (g)
Caloric intake(cal)
b 75.0
b b
70.0 65.0
0.60 d
d
Sham
IO
0.40
60.0
0.20
55.0
0.00
50.0 Sham
IO
Arg 300mg
Arg 600mg
Impact®
Figure 1 Daily caloric intake. Data are expressed as mean 7 SD (n ¼ 8). Different letters indicate statistically significant difference.
b
c
0.80
Arg 300mg
Arg 600mg
Impact®
Figure 4 Daily arginine intake. Data are expressed as mean 7 SD (n ¼ 8). Different letters indicate statistically significant difference. a 60.0
8.0 a b
50.0
b
6.0
Weightgain (g)
Protein intake (g)
7.0
a
b
5.0 4.0
40.0 30.0 20.0
3.0 10.0
2.0 1.0
0.0 Sham
0.0 Sham
IO
Arg 300mg
Arg 600mg
®
Impact
Figure 2 Daily protein intake. Data are expressed as mean 7 SD (n ¼ 8). Different letters indicate statistically significant difference. 1.40
Nitrogen intake (g)
1.20 1.00
b c
Arg 300mg
Arg 600mg
Impact®
Weight gain. Data are expressed as mean 7 SD
differences, weight gain was similar amongst the groups (p40.05; Fig. 5).
Biodistribution of 99mTc-E. coli and effects of arginine on bacterial translocation
a
c c
0.80 0.60 0.40 0.20 0.00 Sham
Figure 5 (n ¼ 8).
IO
IO
600mg Arg
300mg Arg
The uptake of 99mTc-E. coli by blood, MLN, liver, spleen and lungs of the animals in the IO group was significantly higher than that in the sham group (po0.05; Table 2). On the other hand, the administration of arginine (300 mg/day, 600 mg/ day or present in Impacts) reduced the level of bacterial translocation to the blood and all organs investigated despite the fact that all these animals had also undergone intestinal obstruction and inoculation of bacteria (po0.05; Table 2).
Impact®
Figure 3 Daily nitrogen intake. Data are expressed as mean 7 SD (n ¼ 8). Different letters indicate statistically significant difference.
Discussion The role of arginine in preventing bacterial translocation in an experimental intestinal obstruction model was assessed.
ARTICLE IN PRESS Arginine and bacterial translocation
Table 2
Biodistribution of
339
99m
Tc-E. coli (cpm/g).
Organ/blood
Sham
IO
Arg 300 mg/day
Arg 600 mg/day
Impacts
Blood MLN Spleen Liver Lung
92.86** 104.88** 153.11** 177.32** 74.68*
2128,57* 964,28* 742,47* 1144,19* 613,15*
150.00** 45.86** 108.62** 238.21** 55.35**
68.75** 193.98** 132.55** 226.21** 121.38**
250.00** 187.94** 82.04** 189.68** 59.30**
Simultaneous comparison was done among the different groups of treatments. Data are expressed as median (n ¼ 8). cpm, counts per minute. IO group: animals fed conventional chow diet, submitted to intestinal obstruction. * po0.05; **NS (p40.05).
Although arginine has been reported to reduce bacterial translocation in experimental obstructive jaundice models by recovering viable organisms from the mesenteric lymph node (MLN) and spleen16–18 in our study we used 99mTechnetium E. coli. This method is very fast, direct, simple and does not require aseptic conditions throughout the experiments.13,15 Another key point in the present study was that pure arginine or present in an enteral formula was added to conventional chow diet and given daily by regular oral route to the animals, instead of fed by gavage. This was done because in the pilot study, it was observed that gavage fed animals presented with episodes of regurgitation which could not guarantee the exact calculated and individual amount of arginine to be supplemented, and therefore could jeopardize the final outcome. As a consequence, there was a difference in the caloric, protein, nitrogen and arginine ingestion amongst the different supplemented groups (Figs. 1–4). Protein and therefore nitrogen intake were higher in Arg 300 mg and Arg 600 mg groups because of increased protein/ nitrogen content of the supplemented chow with arginine. However, when assessing arginine intake, this was increased in all the three supplemented diet fed animals when compared to the conventional chow groups, with arginine intake significantly higher in Arg 600 mg group (Fig. 4). However, these differences did not impact on body weight (Fig. 5) and on the overall uptake of 99mTechnetium E. coli amongst the three supplemented diets (Table 2). In fact, higher doses of protein/nitrogen and of arginine, as seen in the 600 mg and the Impacts groups, did not lead to a more significant decrease on bacterial translocation, pointing to the advantage of probably using smaller doses of arginine. Bacterial translocation in the supplemented groups was similar to the sham operated animals. According to Macfie et al.19 and Lichitman et al.,20 bacterial translocation is a physiologic phenomenon needed to the development of the gastrointestinal immune system. Our results are in accordance with this finding, since we observed radioactivity in sham group organs. In the present study, translocation was significantly increased in the obstruction group without arginine treatment (IO) indicating that intestinal obstruction really leads to an increase of bacteria translocation. In this situation, the probable involved mechanism is physical disruption of the gut mucosal barrier by direct injury to the enterocytes and its junctions or by decrease of intestinal blood flow.
Arg 300 mg/day, Arg 600 mg/day and Impacts groups showed significant decreased of uptake of 99mTc-E. coli to all organs and blood when compared with IO group. Furthermore, arginine decreased translocation to levels similar to the sham operated group. These findings indicate that arginine was able to reduce bacterial translocation to physiological levels. Previous studies have demonstrated that arginine may improve mucosal morphometric aspects and reduce bacterial translocation in stress situations4,7,21 such as in endotoxemia induced by intestinal obstruction,22 similar to the model used in this study, by increasing villous height and by also attenuating mucosal intestinal injury. These effects of arginine on intestinal mucosal may be due to its direct action on wound healing, collagen deposition, nitrogen retention and polyamine synthesis.4,7 The increase of IgA secretion might also be the mechanism of action induced by arginine to help reduce bacteria translocation. Secretory IgA is an important effector of specific immunity against intraluminal pathogens binding to the surface antigens of invading pathogens and preventing their attachment to the mucosal surface.23 In an experimental model of septic peritonitis, arginine supplementation tended to enhance intestinal IgA secretion.8 However, contrary to the previous reports of a protective role of arginine, Samel et al.9 observed that intravenous infusion of arginine accelerated bacterial translocation to the submucosa and the muscularis propria. Their findings suggest that exogenous arginine and a subsequent increase of endogenous nitric oxide may have a negative effect on intestinal barrier function during small bowel obstruction. According to the authors the probable mechanism was the blockage of inducible NOS which has been found to release far greater amounts of NO and its activation is more likely to be responsible for tissue injury. Therefore, future studies are demanded in order to assess through which pathway arginine plays its role on bacterial translocation, elucidating thus the controversial results. In conclusion, the results of this study showed that arginine, in concentrations of 300 mg/day, 600 mg/day or present in the enteral formula Impacts, was capable of reducing bacterial translocation to physiological levels in an intestinal obstruction model. Future studies are necessary to better understand the mechanism of action of arginine on bacterial translocation, specially in order to be able to translate such results from experimental models into clinical studies and practice.
ARTICLE IN PRESS 340
Acknowledgments Ajionomoto, Brazil and Novartis Brazil for having, respectively, given Arginine and the Impact formula. CNPQ (National Counsel of Technological and Scientific Development), Fundep (Fundac- a ˜o de Desenvolvimento da Pesquisa) for the grants. Iara Eliza Pacı´fico Quirino contributed the conception and design of the experiment and experimental procedures, statistical analysis and interpretation of data; Maria Isabel T.D. Correia the conception and design of the study, interpretation of the data, revision and final approval of the manuscript; and Valbert Nascimento Cardoso the conception and design of the study, quality control of the experimental model and revision of the manuscript.
References 1. Zaloga GP. Immune-enhancing enteral diets: where’s the beef? Crit Care Med 1998;26:1143–6. 2. O’Callaghan G, Beale RJ. The role of immune-enhancing diets in the management of perioperative patients. Crit Care Resusc 2003;5:277–83. 3. Grimble RF. Immunonutrition. Curr Opin Gastroenterol 2005; 21:216–22. 4. Gurbuz A, Kunzelman J, Ratzer EE. Supplemental dietary arginine accelerates intestinal mucosal regeneration and enhances bacterial clearance following radiation enteritis in rats. J Surg Res 1998;74:149–54. 5. Ochoa JB, Strange J, Kearney P, Gellin G, Endean E, Fitzpatrick E. Effects of L-arginine on the proliferation of T lymphocyte subpopulations. J Parenter Enteral Nutr 2001;25:23–9. 6. Ochoa JB, Makarenkova V, Bansal V. A rational use of immune enhancing diets: when should we use dietary arginine supplementation. Nutr Clin Pract 2004;19:216–25. 7. Ersin S, Tuncyurek P, Esassolak M, et al. The prophylactic and therapeutic effects of glutamine and arginine enriched diets on radiation induced enteritis in rats. J Surg Res 2000;89:121–5. 8. Shang HF, Wang YY, Lai YN, Chiu WC, Yeh SL. Effects of arginine supplementation on mucosal immunity in rats with septic peritonits. Clin Nutr 2004;23:561–9. 9. Samel S, Keese M, Laning S, et al. Supplementation and inhibition of nitric oxide synthesis influences bacterial transit time during bacterial translocation in rats. Shock 2003; 19:378–82.
I.E.P. Quirino et al. 10. Ogata Y, Nishi M, Nakayama H, Kuwahara T, Ohnishi Y, Tashiro S. Role of bile in intestinal barrier function and its inhibitory effect on bacterial translocation in obstructive jaundice in rats. J Surg Res 2002;115:18–23. 11. De Oca J, Millat E, Dominguez MA, Aldeano A, Martin R. Selective bowel decontamination, nutritional therapy and bacterial translocation after burn injury. Clin Nutr 1993; 12:355–9. 12. Wiest R, Rath HC. Bacterial translocation in the gut. Best Pract Res Clin Gastroenterol 2003;17:397–425. 13. Diniz SOF, Barbosa AJA, Durval-Arau ´jo I, Lee-Nelson D, Machado LAS, Cardoso VN. Assessment of bacterial translocation in obstructive jaundice using 99mTc-Escherichia coli. Br Arch Biol Technol 2005;48:45–9. 14. Salvallagio PRO, Neto CZ, Tolazzi ARD, Gasparetto EL, Coelho JCU, Campos ACL. Oral glutamine does not prevent bacterial translocation in rats subjected to intestinal obstruction and Escherichia coli challenge but reduces systemic bacteria spread. Nutrition 2002;18:334–7. 15. Oliveira MA, Lemos DS, Diniz SO, Coelho JV, Cardoso VN. Prevention of bacterial translocation using glutamine: a new strategy of investigation. Nutrition 2006;22:419–24. 16. Kuru B, Dinc S, Altinok G, Camlibel M, Gulcelik MA, Alagol H. Effect of different enteral nutrients on bacterial translocation in experimental obstructive jaundice. Eur Surg Res 2004; 36:45–52. 17. Adjei AA, Yamauchi K, Nakasone Y, Konishi M, Yamamoto S. Arginine-supplemented diets inhibit endotoxin-induced bacterial translocation in mice. Nutrition 1995;11:371–4. 18. Zulfikaroglu B, Zulfikaroglu E, Ozmen MM, et al. The effect of immunonutrition on bacterial translocation, and intestinal villus atrophy in experimental obstructive jaundice. Clin Nutr 2003;22:277–81. 19. MacFie J. Enteral versus parenteral nutrition: the significance of bacterial translocation and gut-barrier function. Nutrition 2000;16:606–11. 20. Lichtman SM. Bacterial translocation in humans. J Pediatr Gastroenterol Nutr 2001;33:1–10. 21. Wang JY, Song WL, Zhang LH. Effect of arginine on gastrointestinal immunity during total parenteral nutrition. Clin Nutr 1996;15:115–8. 22. Sukhotnik I, Mogilner J, Krausz MM, et al. Oral arginine reduces gut mucosal injury caused by lipopolysaccharide in rat. J Surg Res 2004;122:256–62. 23. Sawai T, Goldstone N, Drongowski RA. Effect of secretory immunoglobulin A on bacterial translocation in an enterocyte–lymphocyte co-culture model. Pediatr Surg Int 2001;17:275–9.
Available at www.sciencedirect.com
http://intl.elsevierhealth.com/journals/clnu
ORIGINAL ARTICLE
The impact of arginine on bacterial translocation in an intestinal obstruction model in rats Iara Eliza Pacı´fico Quirinoa, Maria Isabel Toulson Davisson Correiab,, Valbert Nascimento Cardosoa a
School of Pharmacy, Federal University of Minas Gerais, Brazil School of Medicine, Federal University of Minas Gerais, Av. Carandaı´ 246 apt. 902, Belo Horizonte, MG 30130-060, Brazil
b
Received 10 July 2006; accepted 19 December 2006
KEYWORDS Arginine; Bacterial translocation; Morbidity; Mortality
Summary Background & aims: Arginine has been shown to have multiple beneficial metabolic and immunologic effects in stress situations. Supplementation of arginine has been shown to promote wound healing and intestinal mucosal recovery after trauma, ischemia or intestinal resection. Bacterial translocation has also been evaluated although with conflicting results and using different assessing techniques. Therefore, the aim of this study was to evaluate the effects of arginine on bacterial translocation in an intestinal obstruction model in rats using Escherichia coli labeled with 99mTechnetium. Methods: Male Wistar rats (250–350 g) were randomized to receive conventional chow, diet supplemented with pure arginine or diet supplemented with an immunonutrition enteral formula, enriched with arginine, omega-3 fatty acid and RNA. After 7 days, the animals were anesthetized. Terminal ileum was isolated and a ligature was placed around it. E. coli labeled with 99mTechnetium (99mTc-E. coli) was inoculated into the intestinal lumen (terminal ileum). After 24 h, the animals were sacrificed. Blood, mesenteric lymph nodes (MLN), liver, spleen and lungs were removed for radioactivity determination. Results: Arginine supplementation (300 mg/day, 600 mg/day or present in the enteral formula) reduced the level of bacterial translocation when compared with the control group (po0.05). This was shown by significantly decrease uptake of 99mTc-E. coli in blood, MLN, liver, spleen and lungs of the animals in the experimental groups (po0.05).
Corresponding author. Tel.: +55 31 91688239; fax: +55 31 32411367.
E-mail addresses: [email protected] (I.E.P. Quirino), [email protected] (M.I.T.D. Correia), [email protected] (V.N. Cardoso). 0261-5614/$ - see front matter & 2007 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved. doi:10.1016/j.clnu.2006.12.007
ARTICLE IN PRESS 336
I.E.P. Quirino et al. Conclusions: These results have shown that arginine was able to decrease bacteria translocation despite intestinal obstruction. There are several mechanisms which might explain the role of arginine and these will be the subject of future studies. & 2007 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.
Background Nutrition has usually been seen as the supply of energy and nitrogen to maintain body mass. Recent studies have, however, revealed that nutrition also plays an important role in modulating the immune and inflammatory responses. The clinical relevance of this modulation has led to the investigation of diets containing specific substrates the socalled immunonutrients, such as glutamine, arginine, nucleotides and omega-3 fatty acids.1 These formulas have been related to modulation of gut function, reduction of infectious complications as well as hospital stay and ventilator days in critically ill patients.2,3 However, because these diets contain more than one immunonutrient, it is difficult to determine which of these elements plays the key role in modulating such phenomena. Thus, attempts to assess the individual role of each of them in different settings are crucial. Arginine, a semiessential amino acid, has been shown to have multiple beneficial metabolic and immunologic effects, especially in stress situations. It seems to promote nitrogen retention and wound healing in the form of increased wound tensile strength and reparative collagen deposition.4 Arginine is important for physiologic functions of immune cells. Ochoa et al.5 observed that T lymphocytes grown in arginine deficient media exhibited decreased membrane expression of T-cell receptors and deficient phenotypic differentiation toward the formation of memory T-cells. Arginine is also necessary for the normal function of myeloid cells such as macrophages and dendritic cells. Myeloid cell activation, such as in critical conditions, induces the expression of membrane arginine transport mechanism and increases arginine uptake. The products of arginine’s metabolism are polyamines (via arginase) and nitric oxide (via nitric oxide synthase). Polyamines such as putrescine, spermine and spermidine act in membrane transport, cell growth, cell proliferation and cell differentiation. Nitric oxide is a vasodilator and an antimicrobial agent effective against intracellular pathogens, extracellular parasites and bacteria. The disease process will determine the metabolic pathways and the ultimate metabolic fate of arginine in the immune system.6 Some studies have shown that arginine decreases bacteria translocation in radiation enteritis and peritonitis animal models whose diets were supplemented with it.4,7,8 However, Samel et al.9 observed that when arginine was given intravenously, bacterial translocation was increased. The exact role exerted by arginine in the process of bacterial translocation is still unclear. This fact may be due to the many mechanisms enrolled in bacterial translocation associated to different critical situations such as hemorrhagic shock, burns, malnutrition, intestinal obstruction, cirrhosis, jaundice, acute pancreatitis, prolonged use of antibiotics,
total parenteral nutrition and magnitude of the operation. Ischemia, increase of intestinal permeability and overgrowth of enteric bacteria are observed in these conditions and result in the spread of bacteria from the gut to other organs.4,10–12 Translocation of bacteria and endotoxins across a leaky mucosa may be responsible for the release of systemic mediators and activation of immunologic cells that contribute to the development of systemic inflammation and multiple organ failure. This is a major cause of death in critically ill patients.1,6,12 In the present study, the role of arginine on bacterial translocation from the intestinal lumen to sterile organs such as liver, spleen, lungs and mesenteric lymph nodes in an intestinal obstruction model assessed by Escherichia coli labeled with 99mTechnetium was investigated.
Methods Animals Forty male Wistar rats weighting between 250 and 350 g were included in this study. The animals were randomized to five groups of eight animals each one: (1) ‘‘sham’’ operated (no obstruction; inoculation of 12.5 MBq of 99mTechnetiumEscherichia coli (99mTc-E. coli)) fed a conventional chow diet (LABINAs, Purina, Brazil; 23% protein); (2) IO group fed a conventional chow diet, submitted to intestinal obstruction and inoculation of 12.5 MBq of 99mTc-E. coli; (3) Arginine 300 mg group fed conventional chow supplemented with arginine 300 mg/day (L-arginine, Ajinomoto do Brazil, Sa ˜o Paulo, Brazil), submitted to intestinal obstruction and inoculation of 12.5 MBq 99mTc-E. coli; (4) Arginine 600 mg group fed conventional chow supplemented with Arginine 600 mg/day, submitted to intestinal obstruction and inoculation of 12.5 MBq of 99mTc-E. coli; (5) Impacts group fed conventional chow supplemented with enteral formula Impacts (Novartis, Rio de Janeiro, Brazil; 22% protein) amounting 600 mg/day of arginine, submitted to intestinal obstruction and inoculation of 99mTc-E. coli. The animals were fed these diets for 7 days previous to intestinal injury and were monitored daily in terms of ingestion and body weight. The supplemented diets were offered first and only when they were finished were the animals allowed conventional chow ad libitum. Water was given ad libitum. The research protocol was approved by the Ethical Committee of Animal Research of Federal University of Minas Gerais.
Preparation of the supplemented diet Conventional rat chow was ground to powder (89%) and added to arginine powder (6%), colorless gelatin powder (4%), corn
ARTICLE IN PRESS Arginine and bacterial translocation starch (1%) and water. Chow powder and arginine powder were homogenized with dissolved gelatin (warm water was used in this process) and extra water was added to guarantee the homogenization. The homogenized diet mass was then divided in small cylinders (pellets). These cylinders were dried in an oven at 45 1C for 8 h. For the Impacts supplemented diet, conventional chow powder (38.1%) was added to the enteral formula (61.9%) and water. The percentage of carbohydrates was derived by the following equation: carbohydrate ¼ total solids (protein+fat+ashes). It was considered that 100 g of protein was equal to 16 g of nitrogen.
Radiolabeling of E. coli A sample of E. coli ATCC-10536 culture grown overnight in tripticasein agar was transferred to 10 mL of saline solution. Bacterial concentration of 108 CFU/mL was adjusted spectophotometrically in 31% of transmittance in 580 nm. An aliquot of bacterial solution (2 mL) was incubated in tubes with 1 mL of stannous chloride solution (580 mM, pH 7.0) at 37 1C for 10 min. After incubation, 1.0–1.5 mCi of 99mTc were added and the preparation was kept at 37 1C for 10 min. The tubes were then centrifuged at 3000 g for 25 min and 100 mL of the supernatant and 100 mL of re-suspended precipitate in saline were used to measure radioactivity. This procedure was repeated three times. The 99mTc percent incorporated into bacterial cells was determined using the following equation13: % labeled bacteria cpm of precipitate 100, ¼ cmp of precipitate þ cpm of supernatant where cpm is the counts per minute.
Surgical procedure Seven days after the beginning of the study, the animals were anesthetized intraperioneally with tiazine (8.7 mg/kg) and ketamine (25.2 mg/kg) solutions. The abdomen was opened through a midline incision and the terminal ileum was isolated and ligated.14 The solution with labeled 99mTcE. coli was inoculated into the distal ileum. The abdominal wound was closed in two layers.
Bacterial translocation analysis Twenty-four hours later, the animals were once again anesthetized using the same technique. Blood (7 mL), Table 1
337 mesenteric lymph nodes (MLN), liver, spleen and lungs were removed, weighed, washed and placed in tubes for determination of radioactivity.15
Statistical analysis Bacterial translocation: statistical analysis among the groups was performed using the Kruskall–Wallis analysis of variance and a post hoc analysis using the Dunn’s test. Data are expressed as median. Differences were considered statistically significant when p values were less than 0.05. Weight, caloric, protein, nitrogen and arginine intake were compared using the ANOVA test and a post hoc analysis using the Duncan’s test. Data were expressed as mean7 standard deviation (SD). Differences were considered statistically significant when p values were less than 0.05. All analysis were performed using the program ‘‘BioEstat Version 3.0 (Mamiraua ´ Civil Society/MCT – CNPq)’’.
Results Composition of standard chow and supplemented chows The composition of standard and supplemented chows is shown in Table 1. There was an increase in total calories in the Impact formula (355.6 kcal/100 g) compared to the conventional chow (275 kcal/100 g) and the arginine supplemented chow (264.3 kcal/100 g). There was an increase of protein, nitrogen and arginine in the chow supplemented with arginine and an increase of arginine, fat and total calories in the chow supplemented with the enteral formula. Carbohydrate content was slight reduced in all supplemented diets when compared to the standard chow.
Caloric ingestion, protein ingestion and weight gain The Impacts group had higher caloric ingestion (Fig. 1) than the other groups (po0.05). Protein and nitrogen intake (Figs. 2 and 3) were higher in Arginine 300 mg and Arginine 600 mg groups (po0.05). Arginine 600 mg group showed higher arginine intake, followed by Impacts and Arginine 300 mg groups (Fig. 4). Arginine represented 1.8% of the total caloric value in the Arginine 300 mg group, 3.3% in the 600 mg and 3.1% in the Impact group. Despite these
Composition of standard chow and supplemented diets (percents).
Nutrients/calories
Standard chow
Supplemented chow with arginine
Supplemented chow with enteral formula
Carbohydrates Fat Protein Nitrogen Arginine Water, ashes and fiber
63.0 4.0 23.0 3.7 1.5 10.0
56.1 3.5 28.1 4.5 6.6 13.3
56.9 8.2 22.9 3.7 3.7 12.0
ARTICLE IN PRESS 338
I.E.P. Quirino et al. 1.20
85.0 ab
80.0
a
a 1.00 Arginine intake (g)
Caloric intake(cal)
b 75.0
b b
70.0 65.0
0.60 d
d
Sham
IO
0.40
60.0
0.20
55.0
0.00
50.0 Sham
IO
Arg 300mg
Arg 600mg
Impact®
Figure 1 Daily caloric intake. Data are expressed as mean 7 SD (n ¼ 8). Different letters indicate statistically significant difference.
b
c
0.80
Arg 300mg
Arg 600mg
Impact®
Figure 4 Daily arginine intake. Data are expressed as mean 7 SD (n ¼ 8). Different letters indicate statistically significant difference. a 60.0
8.0 a b
50.0
b
6.0
Weightgain (g)
Protein intake (g)
7.0
a
b
5.0 4.0
40.0 30.0 20.0
3.0 10.0
2.0 1.0
0.0 Sham
0.0 Sham
IO
Arg 300mg
Arg 600mg
®
Impact
Figure 2 Daily protein intake. Data are expressed as mean 7 SD (n ¼ 8). Different letters indicate statistically significant difference. 1.40
Nitrogen intake (g)
1.20 1.00
b c
Arg 300mg
Arg 600mg
Impact®
Weight gain. Data are expressed as mean 7 SD
differences, weight gain was similar amongst the groups (p40.05; Fig. 5).
Biodistribution of 99mTc-E. coli and effects of arginine on bacterial translocation
a
c c
0.80 0.60 0.40 0.20 0.00 Sham
Figure 5 (n ¼ 8).
IO
IO
600mg Arg
300mg Arg
The uptake of 99mTc-E. coli by blood, MLN, liver, spleen and lungs of the animals in the IO group was significantly higher than that in the sham group (po0.05; Table 2). On the other hand, the administration of arginine (300 mg/day, 600 mg/ day or present in Impacts) reduced the level of bacterial translocation to the blood and all organs investigated despite the fact that all these animals had also undergone intestinal obstruction and inoculation of bacteria (po0.05; Table 2).
Impact®
Figure 3 Daily nitrogen intake. Data are expressed as mean 7 SD (n ¼ 8). Different letters indicate statistically significant difference.
Discussion The role of arginine in preventing bacterial translocation in an experimental intestinal obstruction model was assessed.
ARTICLE IN PRESS Arginine and bacterial translocation
Table 2
Biodistribution of
339
99m
Tc-E. coli (cpm/g).
Organ/blood
Sham
IO
Arg 300 mg/day
Arg 600 mg/day
Impacts
Blood MLN Spleen Liver Lung
92.86** 104.88** 153.11** 177.32** 74.68*
2128,57* 964,28* 742,47* 1144,19* 613,15*
150.00** 45.86** 108.62** 238.21** 55.35**
68.75** 193.98** 132.55** 226.21** 121.38**
250.00** 187.94** 82.04** 189.68** 59.30**
Simultaneous comparison was done among the different groups of treatments. Data are expressed as median (n ¼ 8). cpm, counts per minute. IO group: animals fed conventional chow diet, submitted to intestinal obstruction. * po0.05; **NS (p40.05).
Although arginine has been reported to reduce bacterial translocation in experimental obstructive jaundice models by recovering viable organisms from the mesenteric lymph node (MLN) and spleen16–18 in our study we used 99mTechnetium E. coli. This method is very fast, direct, simple and does not require aseptic conditions throughout the experiments.13,15 Another key point in the present study was that pure arginine or present in an enteral formula was added to conventional chow diet and given daily by regular oral route to the animals, instead of fed by gavage. This was done because in the pilot study, it was observed that gavage fed animals presented with episodes of regurgitation which could not guarantee the exact calculated and individual amount of arginine to be supplemented, and therefore could jeopardize the final outcome. As a consequence, there was a difference in the caloric, protein, nitrogen and arginine ingestion amongst the different supplemented groups (Figs. 1–4). Protein and therefore nitrogen intake were higher in Arg 300 mg and Arg 600 mg groups because of increased protein/ nitrogen content of the supplemented chow with arginine. However, when assessing arginine intake, this was increased in all the three supplemented diet fed animals when compared to the conventional chow groups, with arginine intake significantly higher in Arg 600 mg group (Fig. 4). However, these differences did not impact on body weight (Fig. 5) and on the overall uptake of 99mTechnetium E. coli amongst the three supplemented diets (Table 2). In fact, higher doses of protein/nitrogen and of arginine, as seen in the 600 mg and the Impacts groups, did not lead to a more significant decrease on bacterial translocation, pointing to the advantage of probably using smaller doses of arginine. Bacterial translocation in the supplemented groups was similar to the sham operated animals. According to Macfie et al.19 and Lichitman et al.,20 bacterial translocation is a physiologic phenomenon needed to the development of the gastrointestinal immune system. Our results are in accordance with this finding, since we observed radioactivity in sham group organs. In the present study, translocation was significantly increased in the obstruction group without arginine treatment (IO) indicating that intestinal obstruction really leads to an increase of bacteria translocation. In this situation, the probable involved mechanism is physical disruption of the gut mucosal barrier by direct injury to the enterocytes and its junctions or by decrease of intestinal blood flow.
Arg 300 mg/day, Arg 600 mg/day and Impacts groups showed significant decreased of uptake of 99mTc-E. coli to all organs and blood when compared with IO group. Furthermore, arginine decreased translocation to levels similar to the sham operated group. These findings indicate that arginine was able to reduce bacterial translocation to physiological levels. Previous studies have demonstrated that arginine may improve mucosal morphometric aspects and reduce bacterial translocation in stress situations4,7,21 such as in endotoxemia induced by intestinal obstruction,22 similar to the model used in this study, by increasing villous height and by also attenuating mucosal intestinal injury. These effects of arginine on intestinal mucosal may be due to its direct action on wound healing, collagen deposition, nitrogen retention and polyamine synthesis.4,7 The increase of IgA secretion might also be the mechanism of action induced by arginine to help reduce bacteria translocation. Secretory IgA is an important effector of specific immunity against intraluminal pathogens binding to the surface antigens of invading pathogens and preventing their attachment to the mucosal surface.23 In an experimental model of septic peritonitis, arginine supplementation tended to enhance intestinal IgA secretion.8 However, contrary to the previous reports of a protective role of arginine, Samel et al.9 observed that intravenous infusion of arginine accelerated bacterial translocation to the submucosa and the muscularis propria. Their findings suggest that exogenous arginine and a subsequent increase of endogenous nitric oxide may have a negative effect on intestinal barrier function during small bowel obstruction. According to the authors the probable mechanism was the blockage of inducible NOS which has been found to release far greater amounts of NO and its activation is more likely to be responsible for tissue injury. Therefore, future studies are demanded in order to assess through which pathway arginine plays its role on bacterial translocation, elucidating thus the controversial results. In conclusion, the results of this study showed that arginine, in concentrations of 300 mg/day, 600 mg/day or present in the enteral formula Impacts, was capable of reducing bacterial translocation to physiological levels in an intestinal obstruction model. Future studies are necessary to better understand the mechanism of action of arginine on bacterial translocation, specially in order to be able to translate such results from experimental models into clinical studies and practice.
ARTICLE IN PRESS 340
Acknowledgments Ajionomoto, Brazil and Novartis Brazil for having, respectively, given Arginine and the Impact formula. CNPQ (National Counsel of Technological and Scientific Development), Fundep (Fundac- a ˜o de Desenvolvimento da Pesquisa) for the grants. Iara Eliza Pacı´fico Quirino contributed the conception and design of the experiment and experimental procedures, statistical analysis and interpretation of data; Maria Isabel T.D. Correia the conception and design of the study, interpretation of the data, revision and final approval of the manuscript; and Valbert Nascimento Cardoso the conception and design of the study, quality control of the experimental model and revision of the manuscript.
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