The Effects of Intravenous Glutamine on Bacterial Translocation and Intestinal Morphology in Experimental Pancreatitis

Turk J Med Sci
2008; 38 (2): 127-132
ORIGINAL ARTICLE
© TÜB‹TAK
E-mail: medsci@tubitak.gov.tr
The Effects of Intravenous Glutamine on Bacterial
Translocation and Intestinal Morphology in
Hüseyin BERKEM1
Experimental Pancreatitis
Bülent C. YÜKSEL1
Rukiye BERKEM2
Aim: The aim of this study was to investigate the effects of intravenous glutamine administration on bacterial
S. Yi¤it YILDIZ1
translocation and intestinal morphology in rats with induced acute pancreatitis.
Kubilay GÜNDO?DU1
Materials and Methods: Forty-eight Wistar-Albino rats were divided into four groups (n = 12 for each
group). Pancreatitis was induced with ligation of the main pancreatobiliary duct except in the sham group, in
‹. Hakan ÖZEL1
which only periportal dissection was performed. Rats in the sham and control groups were exposed to
Serdar TOPALO?LU3
standard rat pellet. Total parenteral nutrition (TPN) was administered solely to the pancreatitis + TPN group
Süleyman HENG‹RMEN1
and together with glutamine (Gln) to the pancreatitis + TPN + Gln group. Rats were sacrificed at 48 h after
experiment. Venous blood was obtained for blood culture and biochemistry. Tissue samples were obtained
from liver, spleen, pancreas and lymph nodes from the mesentery for bacterial culture. Histopathologic
examination was performed on tissue sections obtained from the pancreas.
1 First Department of Surgery,
Results: Formation of pancreatitis was demonstrated on microscopic examination. Amylase levels were
Ankara Numune Education and
significantly increased in pancreatitis-induced groups when compared to the sham group (P < 0.05). Bacterial
Research Hospital,
translocation was observed in 1 rat (8%) in the sham group, in 7 rats (58%) in controls, in 8 rats (67%) in
Ankara - TURKEY
2
the pancreatitis + TPN group and in 3 rats (25%) in the pancreatitis + TPN + Gln group. Villus heights and
Department of Microbiology,
numbers were significantly increased in TPN-administered groups compared to controls.
Ankara Education and
Research Hospital,
Conclusions: As Gln supplement in TPN treatment reduced the bacterial translocation and stimulated
Ankara - TURKEY
3
intestinal cell division and replication during the severe pancreatitis model, we suggest that application of Gln
Department of Surgery,
into TPN solutions can reduce possible septic complications associated with pancreatitis.
Karadeniz Technical University,
School of Medicine,
Trabzon - TURKEY
Key Words: Acute pancreatitis, bacterial translocation, glutamine, total parenteral nutrition, experimental
surgery
Deneysel Pankreatitlerde ‹ntestinal Morfoloji ve Bakteriyel Translokasyonda
‹ntravenöz Glutamin Etkileri
Amaç: Bu çal›?man›n amac› bakteriyel translokasyonda i.v. glutamin uygulamas›n›n etkilerini ve ratlardaki
tetiklenmi? akut pankreatitlerde instestinal morfolojiyi ara?t›rmakt›.
Yöntem ve Gereç: 48 adet Wistar-Albino rat (n = 12, her grup için) 4 ayr› gruba bölündü. Pankreatitler sham
grubu d›?›nda, ana pankreatobilier kanal›n ligasyonu ile sa¤land›. Sham gruba ise sadece periportal diseksiyon
uyguland›. Sham grubundaki ve kontrol grubundakiler standart rat yemi verildi. Pankreatitli ve TPN grubuna
yaln›zca TPN uyguland› ve pankreatit + TPN + Gln grubuna Glutamin (Gln) ile birlikte uyguland›. Ratlar›n 48s
Received: May 02, 2006
lik gözlemden sonra hayatlar›na son verildi. Kan kültürleri ve biyokimyalar› için venöz kanlar› al›nd›.
Accepted: February 18, 2007
Karaci¤erden, dalaktan, pankreastan ve mezenter lenf nodlar›nda bakteriyel kültür için doku örnekleri al›nd›.
Pankreastan al›nan doku k›s›mlar›nda ise histopatolojik inceleme yap›ld›.
Bulgular: Pankreatitlerin ?ekilleri, mikroskobik incelemeler ile ortaya konuldu. Sham grupla
Correspondence
kar?›la?t›r›ld›¤›nda (P < 0.05) pankreatit tetiklenmi? grupta amilaz düzeyleri belirgin artm›?t›. Sham grupta
bakteriyel translokasyon 1 ratta (% 8) görülürken, kontrol grubunda 7 (% 58), pankreatit + TPN + grubunda
Hüseyin BERKEM
8 ratta (% 67) ve pankreatitli + TPN + Gln grubunda ise 3 rat da (% 25) görüldü. Kontrol grubu ile
Department of Surgery,
k›yasland›¤›nda villus yükseklikleri ve say›lar› TPN alan grupta bariz artm›?t›.
Ankara Numune Education and
Research Hospital,
Sonuç: TPN tedavisine Gln eklenmesi, bakteriyel translokasyonu azaltt›¤› gibi, intestinal hücre bölünmesini ve
06100 S›hhiye, Ankara - TURKEY
replikasyonunu a¤›r pankreatitlerde stimüle etmektedir, TPN solusyonuna Gln ilavesinin pankreatitte muhtemel
septik komplikasyonlar› önleyebilece¤ini dü?ünmekteyiz.
huseyinberkem@yahoo.com
Anahtar Sözcükler: Akut pankreatit, bakteriel translokasyon, glutamin, TPN, deneysel cerrahi
127

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BERKEM, H et al.
The Effects of IV Glutamine in Experimental Pancreatitis
Turk J Med Sci
Introduction
Leverkusen, Germany) 8 mg/kg intramuscularly.
Pancreatic sepsis is the most common cause of major
Following skin preparation with povidone iodine,
morbidity and mortality associated with acute
laparotomy was performed via midline incision. Portal
elements were dissected but pancreatic duct was not
pancreatitis, with the pathogenesis of such infections
ligated in Group I (sham group). In Groups II, III and IV,
remaining unknown (1). Bacterial translocation (BT) is
retroduodenal
surface
was
explored,
the
known as the passage of viable bacteria or endotoxins
pancreaticobiliary duct (PBD) was ligated with 4-0 silk
from the gut to mesenteric lymph nodes (MLNs) and to
close to its entrance to the duodenum, and acute
other organs, which may commence or exacerbate septic
pancreatitis was induced (1,10). The abdomen was closed
states (2,3).
with 2-0 silk sutures in two layers.
Translocation of organisms from the gastrointestinal
After the operation, feeding regimens were continued
tract to extraintestinal sites is known to be promoted by
according to groups for 48 h. Animals in Group I and II
factors causing systemic insult or bowel injury (4).
were fed with standard chow and tap water. Group III
Several studies have demonstrated that intra-abdominal
was treated with TPN. Those in Group IV were exposed
inflammation during acute pancreatitis promotes BT in
to TPN with Gln (0.75 g/kg/day). TPN solution
the absence of obvious microscopic injury of the intestine
administered to Group III contained 25% dextrose and
(3). Glutamine (Gln) is known as the most significant
4% amino acids, while the nutrition given to Group IV
energy source of enterocytes, and lowers the rate of
contained 25% dextrose, 2% amino acids and 2% Gln.
endotoxemia and translocation by preserving mucosal
Both TPN formulas were arranged as isonitrogenous and
integrity (5-9).
isoenergetic. Therapy was started 6 h after the induction
The aim of this study was to determine the effect of
of pancreatitis, and solutions were administered
intravenously administered Gln on BT and intestinal
continuously via Harvard infusion pumps (A Harvard
mucosal integrity in an experimental model of acute
Bioscience Company, Massachusetts, USA).
pancreatitis in rats.
All animals were sacrificed with an overdose of
intravenous sodium thiopental at postoperative 48 h, and
Materials and Methods
samples from peritoneum, liver, MLNs, spleen and blood
were cultured under aerobic conditions. Biochemical and
Experiments were conducted with adult, male Wistar
histological evaluations were performed. Small bowel
rats weighing from 250 to 300 g. They were housed
sections from terminal ileum were prepared for villus
under constant temperature (22?C) and humidity, with
measurement.
12 h dark/light cycles in Selçuk University, Experimental
Research Laboratory. All studies were carried out under
Testing for Translocation of Bacteria
the guidelines of Selçuk University, Institutional Animal
Blood samples were inoculated in aerobic and
Ethics Committee.
anaerobic BacTec (Becton Dickinson Diagnostic
Animals were randomized into four groups of 12 rats
Instrument Systems; Sparks, MD, USA) pediatric blood
each (Table 1). All animals were fed with standard chow
culture bottles. Bottles were incubated in automated
and tap water before the experiment. All animals in total
blood culture system at 37oC, and growth was observed
parenteral nutrition (TPN) groups (Groups III and IV)
for 7 days. Samples with growth were stained with Gram
were anesthetized with ketamine HCl (Ketalar“) (10
stain and sub-cultured in blood agar and eosin-methylene
mg/kg), and a central venous catheter was placed via
blue (EMB) agar (Bio Mérieux Marcy I’Etoile, France)
right internal jugular cut-down using aseptic technique.
mediums for isolation of microorganisms.
Catheters were protected from the movement of the
Lymph nodes and tissue samples taken from
animals. Animals underwent operation after anesthesia by
peritoneum, liver, spleen and pancreas were placed in
ketamine HCl 10 mg/kg and xylazine (Rhompun, Bayer,
pre-weighted tubes containing 5 ml thioglycolate medium
128

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Vol: 38
No: 2
The Effects of IV Glutamine in Experimental Pancreatitis
April 2008
(Merck
Diagnostica,
Darmstadt,
Germany)
for
serum amylase, aspartate transaminase (AST), and
quantitative culture. Tissue samples were homogenized
alanine transaminase (ALT) levels. Assays were performed
and inoculated in blood agar and EMB agar medium (Bio-
using reagent kits and standard applications.
Merieux, Marcy I’Etoile, France) for aerobic culture.
Statistical Analysis
Plaques were incubated at 37oC for 24-48 h. The
Statistical analysis was performed using SPSS version
organisms grown under these conditions were identified
10.0 for Windows 98. Changes in the groups were
by using the standard microbiologic methods and API
compared with the one-way ANOVA and Fisher’s exact
identification (API 20 E; Bio Mérieux 69280, Marcy
test. Differences between groups were analyzed with the
I’Etoile, France) tests.
Student’s t-test. Differences were considered to be
Histologic Examination
significant if p values were less than 0.05.
The terminal ileum was removed after sacrifice.
Specimens were fixed in 10% formalin in 0.15 M
Results
phosphate buffer (pH = 7.2) and were embedded in
paraffin. They were stained with hematoxylin and eosin,
All of the rats in Groups II, III and IV were found to
and examined under light microscope. Morphometric
have pancreatitis at the second laparotomy, 48 h after
analysis was performed using an eyepiece micrometer.
PBD ligation. Moderate (n = 8/12 in Group II, n = 7/12
Mean villus heights and numbers were measured in each
in Group III and n = 8/12 in Group IV) to severe
specimen.
pancreatitis (n = 4/12 in Group II, n = 5/12 in Group III
and n = 4/12 in Group IV) was observed. In these groups,
In histopathologic examination of the pancreas,
parenchymal inflammation-necrosis, peripancreatic fat
interstitial edema, infiltration of inflammatory cells,
necrosis, various degrees of bile reflux, and peritoneal
hemorrhagic areas, and necrosis were investigated. One
fluid accumulation were observed microscopically and
point was given for each finding, and the severity of
macroscopically. In addition to these findings, serum
pancreatitis was determined by adding the obtained
pancreatic amylase and liver transaminase levels were
points. The overall scores were between 0-4, with 0
increased in pancreatitis-induced groups (Table 1).
representing no pancreatitis, 1: mild pancreatitis, 2-3:
All peritoneal swabs were negative. BT rates in all
moderate pancreatitis, and 4: severe pancreatitis (11).
groups are shown in Table 2. Rats in Group II (acute
Plasma Assays
pancreatitis) and Group III (acute pancreatitis + TPN) had
Venous blood samples were obtained before sacrifice,
significantly higher rates of BT than Group IV (acute
and they were processed using an auto-analyzer (Type
pancreatitis + TPN + Gln). Incidences of BT to the MLNs,
717, Hitachi, Tokyo, Japan) for the measurement of
liver, spleen, and blood in all groups are shown in Table
Table 1. Biochemical findings of experiments.
Groups
Amylase (IU/L)
AST
ALT
I (Sham)
62 ± 18*
80 ± 12*
87 ± 23*
II (Pancreatitis)
3180 ± 965
428 ± 24
388 ± 41
III (Pancreatitis + TPN)
3300 ± 1062
400 ± 32
396 ± 15
IV (Pancreatitis + TPN + Gln)
3207 ± 983
396 ± 30
403 ± 13
*P < 0.05 vs group II, III and IV (one-way ANOVA).
AST: Aspartate transaminase. ALT: Alanine transaminase.
129

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BERKEM, H et al.
The Effects of IV Glutamine in Experimental Pancreatitis
Turk J Med Sci
Table 2. Bacterial translocation ratios of the groups.
Table 3. Bacterial translocation incidences in all groups.
Groups
Ratios
Median (cfu/ml)
Groups
MLN
Liver
Spleen
Pancreas
Blood
I (Sham)
1/12
102
I
1
1
1
1
0
II (Pancreatitis)
7/12
105*
II
7
6
4
4
1
III (Pancreatitis + TPN)
8/12
105*
III
8
7
6
6
3
IV (Pancreatitis + TPN + Gln)
3/12
102
IV
3
2
1
1
0
*P < 0.001 vs controls (Fisher’s exact test)
P < 0.001 vs. controls (Fisher’s exact test).
cfu: Colony forming units.
MLN: Mesenteric lymph nodes.
3. Most of the organisms were translocated to the MLNs
Discussion
in pancreatitis-induced groups. E. coli constituted the
The clinical course of pancreatic sepsis, a rare
leading translocated organism. Proteus mirabilis was
complication seen after acute pancreatitis, is generally
found to be another commonly translocated organism in
devastating (up to 80% mortality) (12). Despite the
the experimental groups (Table 4).
improvements in surgical and radiological approaches for
Mean villus heights and numbers decreased
local control of pancreatic infections or medical treatment
significantly in Group II compared to others (Table 5).
of those patients (TPN, intensive antibiotic treatment,
Those in the TPN-administered groups were not
etc.), the presence of higher morbidity and mortality
significantly different; however, mean villus heights and
rates remains unresolved (12-14).
numbers increased in Gln-enriched TPN-administered
The spectrum of organisms in pancreatic infection
animals compared to TPN-administered rats.
consists of Escherichia, Enterobacter, Enterococcus,
Table 4. Translocated microorganisms.
MLN
Liver
Spleen
Pancreas
Blood
Total
E. coli
11
8
6
6
4
35
Proteus mirabilis
8
7
6
6
1
28
K. pneumoniae
2
2
1
1
6
Salmonella spp
1
1
Total
22
17
13
13
5
70
MLN: Mesenteric lymph nodes.
Table 5. Villus characteristics of groups.
Groups
Villus height (mm)
Villus number/cm
I (Sham)
0.131 ± 0.02
94 ± 4
II (Pancreatitis)
0.043 ± 0.03*
35 ± 2*
III (Pancreatitis + TPN)
0.096 ± 0.008
70 ± 3
IV (Pancreatitis + TPN + Gln)
0.106 ± 0.015
79 ± 6
P < 0.05 vs. other groups (one-way ANOVA).
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Vol: 38
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The Effects of IV Glutamine in Experimental Pancreatitis
April 2008
Pseudomonas, Proteus, Bacteroides or Klebsiella species
pancreatitis necessitates a prolonged period of TPN
(15,16). As in cases with hemorrhagic shock (4) or major
treatment. Absence of intestinal stimulation during TPN
burns (17), the gut is a potential source of pancreatic
treatment promotes BT from the gut (24). To strengthen
sepsis in that enteric microorganisms translocate to
the improving effects of TPN on gut mucosal barrier,
extraintestinal sites without direct manipulation of the
several agents have been examined (growth factors,
gastrointestinal tract (1-3). Disruption of indigenous
trophic gut hormones, and specific nutrients). Gln, an
intestinal microflora, intestinal mucosal damage and
essential substrate for the gut in intestinal stress
alterations in immunity of the host are all considered as
situations, has been well studied for this goal (5,6,8,25).
the principal factors that promote the passage of bacteria
The net effect of Gln on reduction of BT was clearly
across the mucosal barrier (17-19). Similar changes in
observed in our study, as in previous experiments.
bowel mucosa and reticuloendothelial system during
Postoperative survey of animals in this study was
acute pancreatitis may potentially promote translocation
restricted to 48 hours, which was shorter than the period
(1,20). Another hypothesis for BT in acute pancreatitis is
in the previous studies. Despite this limited follow-up, the
intraperitoneal inflammation (2). The mechanisms by
improving effects of Gln supplement were seen on villus
which intraperitoneal inflammation promotes BT have
morphology and translocation rates. The mechanism of
not yet been defined but may involve the release of
Gln on BT remains unclear. Increased apoptosis during
inflammatory mediators or cytokines (2,21). In addition,
the deprivation of Gln and increased villus height and
direct injury to the intestine during intraperitoneal
number during the treatment with Gln support the theory
inflammation may disrupt intestinal barrier function.
that Gln supplies metabolic energy and nucleotide bases
Examination of the possible mechanisms in BT during
required for cell division and replication of intestinal
acute pancreatitis is not the main scope of this study.
mucosa (7,26). Alteration in gut immune function during
However, our data clearly demonstrated translocation of
the Gln treatment -a point not assessed in our study- may
certain Gram-negative species during acute pancreatitis.
be an important pathway for Gln (5,24,27). In addition,
Forty-eight hours after the onset of acute pancreatitis, E.
studies on gut perfusion have also demonstrated that Gln
coli was found to be the leading translocated organism,
increases splanchnic blood supply and mucosal capillary
and bacteria of the indigenous flora were present in the
blood flow in the ascending colon (8,28).
MLN of every culture-positive animal, as reported
In conclusion, when compared to other medical
previously by Runkel et al. (1).
treatment options in acute pancreatitis (selective gut
Restoration of impaired mucosal integrity may be
decontamination, intravenous imipenem treatment, etc.),
hampered by the lack of enteral alimentation during the
Gln-enriched TPN treatment reduces the prevalence of
severe acute pancreatitis episode, which has been shown
pancreatic infections as appropriately as other medical
to play a decisive role in preserving the intestinal barrier
modalities in experimental models (6,29). Our study
function (21-23). The presence of delayed enteral
supports the latter and the inspiring theory proposed by
nutrition secondary to gut paralysis and recurrent
Foitzik et al. (6), that is, the stimulation of intestinal cell
episodes of abdominal pain during severe acute
division and replication with Gln supplement.
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