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Serum citrulline as a diagnostic marker of sepsis-induced intestinal dysfunction
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ORIGINAL ARTICLE
Serum citrulline as a diagnostic marker of sepsis-induced intestinal dysfunction Li-Juan Shen , Yun-Yan Guan ∗, Xi-Ping Wu , Qian Wang , Liang Wang , Tao Xiao , Hai-Rong Wu , Jin-Gui Wang Department of ICU, Wuxi Hospital of traditional Chinese Medicine, Nanjing University of Chinese Medicine Affiliated Wuxi Hospital, No. 8, South West Road, Wuxi 214071, Jiangsu, China
Summary Objective: To investigate the use of citrulline as an indicator for diagnosing septic acute intestinal dysfunction (SAID) in a rat model. Methods: SD rats were divided into three groups: a normal group (A), a model group (B), and a glutamine group (C). Group B was divided into a 36-h group (B1) and a 72-h feeding group (B2). The concentrations of serum citrulline, intestinal fatty acid-binding protein (I-FABP) and intestinal glutamine and histopathological changes were measured. Results: The lengths of the villus and thicknesses of the mucosal layer in groups B1, B2 and C were significantly different from those in group A. Citrulline concentrations in groups B1, B2 and C were lower than in group A; the serum concentrations in group C were significantly greater than in groups B1 and B2. The I-FABP levels of groups B1, B2 and C were higher than group A; I-FABP levels of groups B1 and B2 were higher than group C. Intestinal glutamine levels of groups B1 and B2 were lower than groups A and C. The serum citrulline of group C was negatively correlated with I-FABP and Chiu’s score. Conclusions: Serum citrulline could be used as the diagnostic indicator of SAID. © 2014 Elsevier Masson SAS. All rights reserved.
Introduction Critically ill patients exhibit various degrees of functional damage to the intestinal epithelium because of reduced
∗ Corresponding author. Tel.: +86 510 88859999; fax: +86 510 88859999. E-mail address: [email protected] (Y.-Y. Guan).
splanchnic blood flow, increased mucosal permeability and bacterial translocation [1]. Reduced splanchnic blood flow is also a major concern during the development of multiple organ dysfunction syndrome (MODS) [1]. Currently, there is no simple, accurate and objective method for assessing the intestinal functions of critically ill patients. The most frequently used procedures for measuring enterocyte function in critically ill patients are sugar absorption tests, bomb calorimetry, and tonometry. However, the above methods
http://dx.doi.org/10.1016/j.clinre.2014.10.002 2210-7401/© 2014 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Shen L-J, et al. Serum citrulline as a diagnostic marker of sepsis-induced intestinal dysfunction. Clin Res Hepatol Gastroenterol (2014), http://dx.doi.org/10.1016/j.clinre.2014.10.002
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have some drawbacks and limitations, and there is no gold standard for determining gastrointestinal function [2]. Citrulline is a non-protein amino acid, produced almost entirely by enterocytes from glutamine [3] that could serve as an index reflecting the function and mass of small intestinal epithelial cells [4]. Previous studies on citrulline have focused only on diseases such as short bowel syndrome [5], viral enteritis [4], intestinal transplants [6], intestinal toxicity due to chemotherapy [7], and Crohn’s disease [8]. More recently, there have been a few studies that have focused on critically ill patients [9,10]. Crenn et al. showed that plasma citrulline levels decreased, and were lower when digestive bacterial translocation occurred at the onset of septic shock. The decreased citrulline levels could be a reflection of early acute intestinal dysfunction [11]. There has also been a report showing that glutamine concentrations decrease in the muscles and plasma of critically ill patients [12]. A citrulline generation test (CGT) [13] was developed based on the assumption that functional competent enterocytes are required to convert glutamine to citrulline. The aim of the current study was to investigate the use of citrulline levels as an indicator of septic acute intestinal dysfunction using a rat model.
Materials and methods Experimental animals and environment The Shanghai Super B&K Laboratory Animal Co., Ltd provided 30 male Sprague-Dawley rats (6 months old and weighing 140—160 g). The feeding and experiments were all performed in the laboratory of Nanjing Keygen Technology Development Co., Ltd. For experiments, rats were put into in cages individually, at 20—25 ◦ C, with a humidity of 40—70%, and 12 h light-dark cycles. The experimental animals had free access to food and water. The experiments had been approved by the Animal Care and Use Committee of the Institute of Nanjing University of Chinese Medicine.
Drugs and preparation Lipopolysaccharide (LPS), L-glutamine, and Escherichia coli 055 (B5, No. 2880) were obtained from SIGMA, (St. Louis, MO, USA). PEPTI-2000 variant powder, consisting of short peptides of whey protein, medium-chain triglycerides and maltodextrin, was produced by Nutricia (Zoetermeer, Dutch). LPS was dissolved in saline at a concentration of 0.45 mg/mL. Peptisorb (125 g) was dissolved in 500 mL water. L-glutamine was dissolved in double distilled water at a concentration of 0.375 mg/mL. An I-FABP ELISA kit was obtained from Shanghai Hufeng Chemical Co., Ltd. Freeze-drying agent (Christ, Germany), phenylisothioovyanate (PITC) (Sigma, St. Louis, MO, USA), triethylamine, sodium acetate, ethanol, disodium hydrogen phosphate, phosphoric acid and acetic acid (all analytical grade) were obtained from Sinopharm, (Sinopharm, Shanghai, China). HPLC-grade methanol and acetonitrile were obtained from Merck (Darmstadt, Germany). Citrulline and Glu standards were obtained from Sigma.
Instrumentation Transmission electron microscopy was performed using a JEM-1011 (Japan). Metabolite analyses were done on a 1525 HPLC (Waters, USA), and a 2489 UV detector (Waters, USA), using an XBridge C18 column (5 m, 4.6 × 250 mm), driven by an Empower2 chromatography workstation.
Experimental methods Experimental grouping Each of the 30 rats was designated to a group (for a total of 10 rats per group): a normal group (A); a model group (B), which was subdivided into groups B1 and group B2 with five rats in each subgroup; and a glutamine group (C). Preparation of animal models LPS can cause intestinal mucosal edema, cell necrosis, increased intestinal permeability, and intestinal mucosal barrier damage, eventually leading to mucosal damage [14]. A sepsis model [15] was prepared using lipopolysaccharide (LPS) at a dose of 4.5 mg/kg as described previously by Mercer et al. [16]. Group A rats were injected intraperitoneally with saline, and were fed standard laboratory feed (343.5 kcal/kg) at a rate of 80 kcal/d (Xietong Organism CO., Ltd.) for 72 h. Group B rats were injected intraperitoneally with 0.45 mg/mL LPS solution at a dose of 1 mL/100 g. Three intraperitoneal injections were completed within 10 minutes. Beginning 12 h later, 4 mL of 25.2% peptisorb solution (80 kcal) was orally administrated by gavage five times per day for 36 h (a mean of 7 doses in 36 h) in group B1 or 72 h (a mean of 15 doses in 72 h) in group B2. Group C rats were injected intraperitoneally with LPS as for group A, and gavaged with peptisorb and glutamine (3.75 g/kg/d) for 72 h. Specimen collection Twelve hours after the final feedings, blood was collected from the orbital venous plexus, and rats were sacrificed for pathological examination of the ileum. Blood was centrifuged at 13,000 rpm for 10 minutes; serum was stored at —80 ◦ C until further testing.
Analyses Pathological examination Small intestines were fixed in 10% formalin followed by dehydration, paraffin embedding, and sectioning. The sections were stained with HE [17] and examined microscopically for edema of the villi, degeneration and necrosis of epithelial cells, congestion, edema and inflammatory cell infiltration of intestinal layers. Samples were scored according to the six-point Chiu score [18]: the degree of pathology ranged from mild to serious based on a scale from 1 to 5; no obvious lesions received 0 points. For EM analysis, software was used to measure the length of the villi and mucosal thickness (DX45, Olympus, Japan, DP2-BSW image analysis system). We calculated the ratio of the average length of villi to mucosal thickness of each animal, and the average
Please cite this article in press as: Shen L-J, et al. Serum citrulline as a diagnostic marker of sepsis-induced intestinal dysfunction. Clin Res Hepatol Gastroenterol (2014), http://dx.doi.org/10.1016/j.clinre.2014.10.002
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Figure 1
The Chiu scores were 0, 1 and 2, respectively.
thickness of the mucosal layer. An intergroup analysis was also performed; the examiners were blinded to the protocol.
I-FABP measurement Serum I-FABP concentrations were measured in 0.1 mL samples using ELISA, according to the kit manufacturer’s instructions. A blank well was set as the zero, and the absorbance of each well (OD) was measured at a wavelength of 450 nm. Each determination was performed within 15 minutes of adding the stop solution [19].
Detection of citrulline Serum (0.4 mL) was used for measurement of serum citrulline concentration according to an HPLC method [20]. In brief, after ultrafiltration to remove proteins, filtrate (100 L) was added to derivation agent (200 L; a mixture of isothiocyanate, ultrapure water, triethylamine, and absolute ethanol at a ratio of 1:1:1:7); hexane (400 L) was then added to the mixture. The bottom layer was filtered with a 0.45-m vacuum filter, and 20 L of the filtrate was injected into the HPLC. The conditions for chromatography included a mixture of 25-mM ammonium acetate buffer (pH 6.0) and methanol initially at a 90:10 ratio, then at 85:15 for 8 min, 55:45 for 10 min, and finally 10:90 for 10 minutes. The flow rate was 1.0 mL/min with a constant column temperature of 30 ◦ C. The detection wavelength was 254 nm.
Measurement of intestinal glutamine Ileal tissue (0.1 g) was used for the measurement of intestinal glutamine concentrations by an HPLC method [21]. Ileal tissue was homogenized with saline (0.5 mL) at 4 ◦ C, and was then combined with acetonitrile (0.5 mL). Samples were centrifuged at 2500 rpm at 4 ◦ C for 20 min. An aliquot of the supernatant (40 L) was mixed with derivation agent (40 L; isothiocyanate, ultrapure water, triethylamine, and ethanol at ratio of 1:1:1:7), and then lyophilized to dryness. Diluent (mobile phase A:B = 10:90) was added and the mixture was filtered through a 0.45-m vacuum filter. A 20-L sample of filtrate was used for HPLC. The mobile phase was 25mM phosphate buffer (pH 5.5) and acetonitrile (90:10 ratio) with a flow rate of 1.0 mL/min at a constant temperature of 30 ◦ C. The detection wavelength was 254 nm. A C18 column (250 mm × 4.6 mm, 5 m) was used.
Statistical processing All data were processed with SPSS 15.0 (CABIT Information Technology, Shanghai, China) software, and expressed as mean ± SD. For parametric data, one-way analysis of variance (ANOVA) was used to compare the four groups with respect to continuous variables. When a statistically significant differences were observed among groups, a LSD test or Dunnett’s T3 test was performed. Pearson correlation analysis was used to describe correlation. A P value less than 0.05 was considered as statistical significance.
Results Histopathological results Small intestines of rats in group A were histologically normal in all layers. The Chiu score of the group was 0. In group B1, one rat had cystic sub-epithelium at the top of the villi accompanied by capillary congestion leading to a Chiu score of 1. The other four animals had no abnormalities resulting in Chiu scores of 0. In group B2, there was moderate lifting of the sub-epithelial layer, and separation from the lamina propria in two rats. The central lacteals were expanded leading to Chiu scores of 2. Two rats had sub-epithelium cystic gaps at the top of villi, resulting in Chiu scores of 1. One rat had no significant lesions and a Chiu score of 0. In group C, six rats had mild expansion of the sub-epithelial layer, cystic gaps at the tops of villi accompanied by capillary congestion resulting in Chiu scores of 1. Two rats had central lacteal expansion and Chiu scores of 2. Two rats had no obvious lesions and Chiu scores of 0. The Chiu score is showed in Fig. 1. Intestinal mucosal damage was evident in groups C and B2. The damage in group B2 was less than that in group C (Table 1). There were statistically significant differences in the intestinal villi length and mucosal thickness when groups B1, B2 and C were compared with group A (P < 0.05; Table 2).
Serum citrulline concentrations The serum citrulline concentrations of groups B1, B2 and C were lower than that of group A (P < 0.05). Serum citrulline concentrations of subgroups B1 and B2 were lower than that of group C (P < 0.05; Table 3).
Please cite this article in press as: Shen L-J, et al. Serum citrulline as a diagnostic marker of sepsis-induced intestinal dysfunction. Clin Res Hepatol Gastroenterol (2014), http://dx.doi.org/10.1016/j.clinre.2014.10.002
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Chiu scores.
Group Group Group Group Group
A B1 B2 C
Number of cases
Chiu score
10 5 5 10
0.1 0.2 1.4 1.0
± ± ± ±
0.416 0.447 1.140a,b 0.667a
Note: F = 6.831, P = 0.002. a P < 0.05 versus group A. b P < 0.05 versus group C.
Serum I-FABP concentrations Serum I-FABP concentrations of groups B1, B2 and C were higher than in group A (P < 0.05); I-FABP concentrations of subgroups B1and B2 were higher than in group C (P < 0.05). The I-FABP concentrations in subgroups B1and B2 also were significantly different (Table 4).
Figure 2 Correlation of serum citrulline and I-FABP concentrations in Group C. Data represent mean ± SD, P < 0.01, R = —0.844 for Pearson correlation analysis.
Intestinal glutamine concentrations Intestinal glutamine concentrations in subgroups B1 and B2 were lower than those in groups A and C (P < 0.05). The concentrations in subgroups B1and B2 were also significantly different (Table 5). Correlation analyses In group C, the serum citrulline concentration was negatively correlated with I-FABP concentration (P < 0.01, R = —0.844), (Fig. 2). In group C, the serum citrulline concentration was negatively correlated with intestinal Chiu score (P < 0.01, R = —0.850), (Fig. 3).
Discussion Currently, the intestinal tract is considered the key to MODS, the amplifier of inflammation, and origin of systemic bacteremia and toxemia [1]. However, due to the body’s protective mechanisms, the intestinal tract is also the most easily-damaged organ [22]. In states of low perfusion, the body redistributes systemic blood flow to protect organs, such as the heart and brain, by reducing the blood flow to the limbs and intestinal tract [22]. When intestinal blood perfusion is reduced, gastrointestinal tissue oxygen supply also decreases, leading to potential intestinal tract damage, and ultimately gastrointestinal dysfunction/failure (GIDF) [22].
Table 2 Group Group Group Group Group
A B1 B2 C
Figure 3 Correlation of serum citrulline concentrations with intestinal Chiu scores in Group C. Data represent mean ± SD (P < 0.01, R = —0.850).
GIDF is common in critically ill patients [23]. There is at least one gastrointestinal symptom in nearly 59% of all intensive care unit (ICU) patients, and there is an association with increased mortality [24]. The ability to sustain or improve nutritional status, and maintain intestinal mucosal barrier function, depends on adequate function of intestinal epithelial cells [1]. Sepsis
Villus measurements. Number of cases
Villus length (m)
10 5 5 10
284.628 239.816 260.060 259.583
± ± ± ±
15.4321 15.3801a 30.508a 17.2101a
Mucosal thickness (m) 539.551 430.450 455.820 449.319
± ± ± ±
44.936 39.0591a 50.956a 33.7501a
Villi length:mucosal thickness 0.528 0.557 0.571 0.578
Note: villi length, F = 6.800, P = 0.002; mucosal thickness: F = 11.551, P = 0.001. a P < 0.05 versus group A.
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Serum citrulline levels in SAID Table 3 Group Group Group Group Group
A B1 B2 C
5
Serum citrulline concentrations (mol/L) (¯ x ± s). Number of cases
Serum citrulline (umol/L)
10 5 5 10
72.568 39.039 38.072 66.997
± ± ± ±
4.161 2.213a,b 1.980a,b 6.1751
Note: Blood was collected 12 hours after the final feedings (F = 107.995, P < 0.001). a P < 0.05 versus group A. b P < 0.05 versus group C.
Table 4
Serum I-FABP Concentrations (ng/L) (¯ x ± s).
Group Group Group Group Group
A B1 B2 C
Number of cases
Serum I- FABP (ng/L)
10 5 5 10
1.228 4.990 10.841 3.044
± ± ± ±
0.123 0.417a,b 1.254a,b,c 0.2961
Note: Blood was collected 12 hours after the final feedings (F = 22.295, P < 0.001). a P < 0.05 versus group A. b P < 0.05 versus group C. c P < 0.05 versus group B1.
is caused by infection of systemic inflammatory response syndrome (SIRS). During sepsis, intestinal epithelial cell apoptosis increases, and the number of cells decreases resulting in epithelial cell dysfunction, damage to intestinal mucosa, intestinal flora imbalance, and inhibition of intestinal immune function. This leads to endotoxin release from the lumen into the blood, and causes increased release of inflammatory mediators, further damaging the intestinal mucosa. Translocation of a large number of bacteria and endotoxins causes the body to increase inflammatory mediators, leading to a vicious cycle resulting in MODS [1,25]. Citrulline is involved in the nitric oxide (NO) cycle, including local recycling of citrulline [26]. It is also involved in the urea cycle in the liver [26]. Finally, citrulline affects the intestinal-renal axis wherein glutamine is converted to arginine [3]. Citrulline is mainly generated through the absorption and metabolism of glutamine by epithelial cells of the small intestine. Although other amino acids such as arginine, proline and ornithine are also involved in the generation of citrulline [13], an isotope-based study showed
Table 5 Glutamine concentrations in intestinal tissue (ng/L) (¯ x ± s). Group Group Group Group Group
A B1 B2 C
Number of cases
Intestinal glutamine (ng/L)
10 5 5 10
2.924 1.023 1.651 2.320
± ± ± ±
0.135 0.039a,b 0.036a,b 0.157
Note: Twelve hours after the final feedings, rats were sacrificed for pathological examination of the ileum. F = 302.220, P < 0.001. a P < 0.05 versus group A. b P < 0.05 versus group C.
that, in humans, 80% of citrulline is generated from the metabolism of glutamine [27]. The glutamine in the intestinal tract or arterial blood is converted into citrulline through the glutamate-ornithine pathway of epithelial cells in the small intestine [28,29]. Citrulline is then released into the hepatic portal vein system. In the current study, we found that citrulline levels in the sepsis model group were significantly lower than those of the control group. Our data are in line with those of Crenn et al. [11] who reported a significant decrease in serum citrulline levels when digestive bacterial translocation occurred at the onset of septic shock. The authors stated that the decreased citrulline levels could be a reflection of early acute intestinal dysfunction. Many recent findings have shown that citrulline can reflect acute decreases in the number of intestinal epithelial cells, and can reflect intestinal toxicity of radiation and chemotherapy [7,30]. It can also serve as an early indicator of acute rejection reaction after intestinal transplantation [28]. Following high-dose chemotherapy, decreases in serum citrulline concentrations are associated with bacteremia [31]. Pediatric studies have also confirmed a correlation between the reduction of serum citrulline concentration and mucosal damage [32]. The current data indicate that the I-FABP concentrations were significantly higher in the sepsis model group, leading to a negative correlation with citrulline in group C (P < 0.01, R = —0.844). It was also found that serum citrulline concentration was negatively correlated with intestinal Chiu score (P < 0.01, R = —0.850). A recent study showed that citrulline level could be used as an indicator of intestinal failure in patients with acute pancreatitis [33]. This study found that the serum citrulline concentrations in patients with severe pancreatitis decreased significantly compared to those with mild pancreatitis, and exhibited a negative correlation with IFABP. The I-FABP was clearly correlated with the extent of gastrointestinal disorder, acute physiology and chronic health evaluation II scores (APACHE II), C-reactive protein levels and ICU stay durations. This study confirmed that, in patients with SIRS and hypovolemia-induced clinical severe intestinal disorders, the degree of intestinal dysfunction was associated with the intestinal epithelial cell necrosis, which was reflected by I-FABP. A decrease in serum citrulline concentrations confirmed the reduction of intestinal epithelial cells. However, in the current study, there was no correlation between citrulline and I-FABP in groups B1 and B2. This inconsistency might have been related to the small sample size, and time points selected. Epithelial cells of the small intestine are the main sources of serum citrulline. A reduction of intestinal epithelial cells during ischemia would cause a decrease in serum citrulline levels. During SIRS, dysfunction of intestinal epithelial cells would also lead to a decline in serum citrulline levels. Hietbrink et al. [34] found that SIRS increased intestinal permeability, while the level of I-FABP did not change in endotoxemia patients. This indicates that SIRS caused increases in permeability, but did not directly damage intestinal epithelial cells. Therefore, the number of intestinal epithelial cells did not decrease at that time. There was only intestinal dysfunction, and yet serum citrulline levels still declined. Because the synthesis of intestinal citrulline occurs in the mitochondria of intestinal epithelial cells, the SIRS-induced mitochondrial dysfunction might lead to the
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decline of serum citrulline levels. In the current study, the 36-h subgroup was created in consideration of this situation. The citrulline concentrations decreased at 36 h, while the I-FABP concentrations significantly increased. This inconsistency might have been related to the small sample size, and time points selected. The current study used the citrulline generation test (CGT) [13]. The citrulline metabolic pathway revealed that the above studies could not completely exclude a lack of glutamine, a citrulline metabolic precursor. Glutamine is an essential nutrient for maintaining intestinal mucosal metabolism, structure and function when patients are under stress. There have been some reports that the glutamine concentrations of muscles and plasma in critically ill patients are reduced [12]. This has led to studies of the CGT [13]. The CGT is based on the assumption that functional competent enterocytes are required to convert glutamine to citrulline. Thus, citrulline produced by the intestine from a fixed load of glutamine, through enteral or intravenous administration, could reflect the function of mucosal epithelial cells. Glutamine administered as an alanine-glutamine dipeptide was used so that the test results would not be affected by the amount of basic glutamine storage [1]. The current study used the common nutritional supplement (peptisorb). In group B, Group C received additional glutamine by gavage, and the glutamine concentration was significantly higher than in the Pepti-only group. Thus, the factor of insufficient substrate was excluded; citrulline better reflected the number and function of intestinal epithelial cells. The hepatic circulatory system has no first-pass effect on citrulline. Liver-produced citrulline can be taken up by the kidney and metabolized to arginine [27], and therefore, citrulline levels can be affected by renal function. This could limit the application of citrulline in assessing the functions of small intestinal epithelial cells, because most critically ill patients have various degrees of renal impairment. The current study did not consider the impact of renal function. This will be considered in a future study. However, some studies have found that even if renal failure was present, if the fasting serum citrulline concentration was below the reference value (< 20 mol/L), it could still reflect the function of intestinal epithelial cells [35]. We conclude that serum citrulline levels could reflect the number and function of intestinal epithelial cells, and might be useful as a non-invasive indicator in the diagnosis of acute intestinal dysfunction in critically ill patients. The feasibility of serum citrulline as a common indicator of clinical detection still needs to be confirmed by human studies.
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Disclosure of interest [16]
The authors have not supplied their declaration of conflict of interest.
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7 [28] Wakabayashi Y, Iwashima A, Yamada E, Yamada R. Enzymological evidence for the indispensability of small intestine in the synthesis of arginine from glutamate. II. Nacetylglutamate synthase. Arch Biochem Biophys 1991;291(1): 9—14. [29] Wakabayashi Y, Jones ME. Pyrroline-5-carboxylate synthesis from glutamate by rat intestinal mucosa. J Biol Chem 1983;258(6):3865—72. [30] Wedlake L, McGough C, Hackett C, Thomas K, Blake P, Harrington K, et al. Can biological markers act as non-invasive, sensitive indicators of radiation-induced effects in the gastrointestinal mucosa? Aliment Pharmacol Ther 2008;27(10):980—7, http://dx.doi.org/10.1111/j.1365-2036.2008.03663.x. [31] Herbers AH, Blijlevens NM, Donnelly JP, de Witte TJ. Bacteraemia coincides with low citrulline concentrations after high-dose melphalan in autologous HSCT recipients. Bone Marrow Transplant 2008;42(5):345—9, http://dx.doi.org/10.1038/ bmt.2008.170. [32] van Vliet MJ, Tissing WJ, Rings EH, Koetse HA, Stellaard F, Kamps WA, et al. Citrulline as a marker for chemotherapy induced mucosal barrier injury in pediatric patients. Pediatr Blood Cancer 2009;53(7):1188—94, http://dx.doi.org/ 10.1002/pbc.22210. [33] Pan L, Wang X, Li W, Li N, Li J. The intestinal fatty acid binding protein diagnosing gut dysfunction in acute pancreatitis: a pilot study. Pancreas 2010;39(5):633—8. [34] Hietbrink F, Besselink MG, Renooij W, de Smet MB, Draisma A, van der Hoeven H, et al. Systemic inflammation increases intestinal permeability during experimental human endotoxemia. Shock 2009;32(4):374—8, http://dx.doi.org/10.1097/ SHK.0b013e3181a2bcd6. [35] Crenn P, Coudray-Lucas C, Thuillier F, Cynober L, Messing B. Postabsorptive plasma citrulline concentration is a marker of absorptive enterocyte mass and intestinal failure in humans. Gastroenterology 2000;119(6):1496—505.
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ORIGINAL ARTICLE
Serum citrulline as a diagnostic marker of sepsis-induced intestinal dysfunction Li-Juan Shen , Yun-Yan Guan ∗, Xi-Ping Wu , Qian Wang , Liang Wang , Tao Xiao , Hai-Rong Wu , Jin-Gui Wang Department of ICU, Wuxi Hospital of traditional Chinese Medicine, Nanjing University of Chinese Medicine Affiliated Wuxi Hospital, No. 8, South West Road, Wuxi 214071, Jiangsu, China
Summary Objective: To investigate the use of citrulline as an indicator for diagnosing septic acute intestinal dysfunction (SAID) in a rat model. Methods: SD rats were divided into three groups: a normal group (A), a model group (B), and a glutamine group (C). Group B was divided into a 36-h group (B1) and a 72-h feeding group (B2). The concentrations of serum citrulline, intestinal fatty acid-binding protein (I-FABP) and intestinal glutamine and histopathological changes were measured. Results: The lengths of the villus and thicknesses of the mucosal layer in groups B1, B2 and C were significantly different from those in group A. Citrulline concentrations in groups B1, B2 and C were lower than in group A; the serum concentrations in group C were significantly greater than in groups B1 and B2. The I-FABP levels of groups B1, B2 and C were higher than group A; I-FABP levels of groups B1 and B2 were higher than group C. Intestinal glutamine levels of groups B1 and B2 were lower than groups A and C. The serum citrulline of group C was negatively correlated with I-FABP and Chiu’s score. Conclusions: Serum citrulline could be used as the diagnostic indicator of SAID. © 2014 Elsevier Masson SAS. All rights reserved.
Introduction Critically ill patients exhibit various degrees of functional damage to the intestinal epithelium because of reduced
∗ Corresponding author. Tel.: +86 510 88859999; fax: +86 510 88859999. E-mail address: [email protected] (Y.-Y. Guan).
splanchnic blood flow, increased mucosal permeability and bacterial translocation [1]. Reduced splanchnic blood flow is also a major concern during the development of multiple organ dysfunction syndrome (MODS) [1]. Currently, there is no simple, accurate and objective method for assessing the intestinal functions of critically ill patients. The most frequently used procedures for measuring enterocyte function in critically ill patients are sugar absorption tests, bomb calorimetry, and tonometry. However, the above methods
http://dx.doi.org/10.1016/j.clinre.2014.10.002 2210-7401/© 2014 Elsevier Masson SAS. All rights reserved.
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have some drawbacks and limitations, and there is no gold standard for determining gastrointestinal function [2]. Citrulline is a non-protein amino acid, produced almost entirely by enterocytes from glutamine [3] that could serve as an index reflecting the function and mass of small intestinal epithelial cells [4]. Previous studies on citrulline have focused only on diseases such as short bowel syndrome [5], viral enteritis [4], intestinal transplants [6], intestinal toxicity due to chemotherapy [7], and Crohn’s disease [8]. More recently, there have been a few studies that have focused on critically ill patients [9,10]. Crenn et al. showed that plasma citrulline levels decreased, and were lower when digestive bacterial translocation occurred at the onset of septic shock. The decreased citrulline levels could be a reflection of early acute intestinal dysfunction [11]. There has also been a report showing that glutamine concentrations decrease in the muscles and plasma of critically ill patients [12]. A citrulline generation test (CGT) [13] was developed based on the assumption that functional competent enterocytes are required to convert glutamine to citrulline. The aim of the current study was to investigate the use of citrulline levels as an indicator of septic acute intestinal dysfunction using a rat model.
Materials and methods Experimental animals and environment The Shanghai Super B&K Laboratory Animal Co., Ltd provided 30 male Sprague-Dawley rats (6 months old and weighing 140—160 g). The feeding and experiments were all performed in the laboratory of Nanjing Keygen Technology Development Co., Ltd. For experiments, rats were put into in cages individually, at 20—25 ◦ C, with a humidity of 40—70%, and 12 h light-dark cycles. The experimental animals had free access to food and water. The experiments had been approved by the Animal Care and Use Committee of the Institute of Nanjing University of Chinese Medicine.
Drugs and preparation Lipopolysaccharide (LPS), L-glutamine, and Escherichia coli 055 (B5, No. 2880) were obtained from SIGMA, (St. Louis, MO, USA). PEPTI-2000 variant powder, consisting of short peptides of whey protein, medium-chain triglycerides and maltodextrin, was produced by Nutricia (Zoetermeer, Dutch). LPS was dissolved in saline at a concentration of 0.45 mg/mL. Peptisorb (125 g) was dissolved in 500 mL water. L-glutamine was dissolved in double distilled water at a concentration of 0.375 mg/mL. An I-FABP ELISA kit was obtained from Shanghai Hufeng Chemical Co., Ltd. Freeze-drying agent (Christ, Germany), phenylisothioovyanate (PITC) (Sigma, St. Louis, MO, USA), triethylamine, sodium acetate, ethanol, disodium hydrogen phosphate, phosphoric acid and acetic acid (all analytical grade) were obtained from Sinopharm, (Sinopharm, Shanghai, China). HPLC-grade methanol and acetonitrile were obtained from Merck (Darmstadt, Germany). Citrulline and Glu standards were obtained from Sigma.
Instrumentation Transmission electron microscopy was performed using a JEM-1011 (Japan). Metabolite analyses were done on a 1525 HPLC (Waters, USA), and a 2489 UV detector (Waters, USA), using an XBridge C18 column (5 m, 4.6 × 250 mm), driven by an Empower2 chromatography workstation.
Experimental methods Experimental grouping Each of the 30 rats was designated to a group (for a total of 10 rats per group): a normal group (A); a model group (B), which was subdivided into groups B1 and group B2 with five rats in each subgroup; and a glutamine group (C). Preparation of animal models LPS can cause intestinal mucosal edema, cell necrosis, increased intestinal permeability, and intestinal mucosal barrier damage, eventually leading to mucosal damage [14]. A sepsis model [15] was prepared using lipopolysaccharide (LPS) at a dose of 4.5 mg/kg as described previously by Mercer et al. [16]. Group A rats were injected intraperitoneally with saline, and were fed standard laboratory feed (343.5 kcal/kg) at a rate of 80 kcal/d (Xietong Organism CO., Ltd.) for 72 h. Group B rats were injected intraperitoneally with 0.45 mg/mL LPS solution at a dose of 1 mL/100 g. Three intraperitoneal injections were completed within 10 minutes. Beginning 12 h later, 4 mL of 25.2% peptisorb solution (80 kcal) was orally administrated by gavage five times per day for 36 h (a mean of 7 doses in 36 h) in group B1 or 72 h (a mean of 15 doses in 72 h) in group B2. Group C rats were injected intraperitoneally with LPS as for group A, and gavaged with peptisorb and glutamine (3.75 g/kg/d) for 72 h. Specimen collection Twelve hours after the final feedings, blood was collected from the orbital venous plexus, and rats were sacrificed for pathological examination of the ileum. Blood was centrifuged at 13,000 rpm for 10 minutes; serum was stored at —80 ◦ C until further testing.
Analyses Pathological examination Small intestines were fixed in 10% formalin followed by dehydration, paraffin embedding, and sectioning. The sections were stained with HE [17] and examined microscopically for edema of the villi, degeneration and necrosis of epithelial cells, congestion, edema and inflammatory cell infiltration of intestinal layers. Samples were scored according to the six-point Chiu score [18]: the degree of pathology ranged from mild to serious based on a scale from 1 to 5; no obvious lesions received 0 points. For EM analysis, software was used to measure the length of the villi and mucosal thickness (DX45, Olympus, Japan, DP2-BSW image analysis system). We calculated the ratio of the average length of villi to mucosal thickness of each animal, and the average
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Figure 1
The Chiu scores were 0, 1 and 2, respectively.
thickness of the mucosal layer. An intergroup analysis was also performed; the examiners were blinded to the protocol.
I-FABP measurement Serum I-FABP concentrations were measured in 0.1 mL samples using ELISA, according to the kit manufacturer’s instructions. A blank well was set as the zero, and the absorbance of each well (OD) was measured at a wavelength of 450 nm. Each determination was performed within 15 minutes of adding the stop solution [19].
Detection of citrulline Serum (0.4 mL) was used for measurement of serum citrulline concentration according to an HPLC method [20]. In brief, after ultrafiltration to remove proteins, filtrate (100 L) was added to derivation agent (200 L; a mixture of isothiocyanate, ultrapure water, triethylamine, and absolute ethanol at a ratio of 1:1:1:7); hexane (400 L) was then added to the mixture. The bottom layer was filtered with a 0.45-m vacuum filter, and 20 L of the filtrate was injected into the HPLC. The conditions for chromatography included a mixture of 25-mM ammonium acetate buffer (pH 6.0) and methanol initially at a 90:10 ratio, then at 85:15 for 8 min, 55:45 for 10 min, and finally 10:90 for 10 minutes. The flow rate was 1.0 mL/min with a constant column temperature of 30 ◦ C. The detection wavelength was 254 nm.
Measurement of intestinal glutamine Ileal tissue (0.1 g) was used for the measurement of intestinal glutamine concentrations by an HPLC method [21]. Ileal tissue was homogenized with saline (0.5 mL) at 4 ◦ C, and was then combined with acetonitrile (0.5 mL). Samples were centrifuged at 2500 rpm at 4 ◦ C for 20 min. An aliquot of the supernatant (40 L) was mixed with derivation agent (40 L; isothiocyanate, ultrapure water, triethylamine, and ethanol at ratio of 1:1:1:7), and then lyophilized to dryness. Diluent (mobile phase A:B = 10:90) was added and the mixture was filtered through a 0.45-m vacuum filter. A 20-L sample of filtrate was used for HPLC. The mobile phase was 25mM phosphate buffer (pH 5.5) and acetonitrile (90:10 ratio) with a flow rate of 1.0 mL/min at a constant temperature of 30 ◦ C. The detection wavelength was 254 nm. A C18 column (250 mm × 4.6 mm, 5 m) was used.
Statistical processing All data were processed with SPSS 15.0 (CABIT Information Technology, Shanghai, China) software, and expressed as mean ± SD. For parametric data, one-way analysis of variance (ANOVA) was used to compare the four groups with respect to continuous variables. When a statistically significant differences were observed among groups, a LSD test or Dunnett’s T3 test was performed. Pearson correlation analysis was used to describe correlation. A P value less than 0.05 was considered as statistical significance.
Results Histopathological results Small intestines of rats in group A were histologically normal in all layers. The Chiu score of the group was 0. In group B1, one rat had cystic sub-epithelium at the top of the villi accompanied by capillary congestion leading to a Chiu score of 1. The other four animals had no abnormalities resulting in Chiu scores of 0. In group B2, there was moderate lifting of the sub-epithelial layer, and separation from the lamina propria in two rats. The central lacteals were expanded leading to Chiu scores of 2. Two rats had sub-epithelium cystic gaps at the top of villi, resulting in Chiu scores of 1. One rat had no significant lesions and a Chiu score of 0. In group C, six rats had mild expansion of the sub-epithelial layer, cystic gaps at the tops of villi accompanied by capillary congestion resulting in Chiu scores of 1. Two rats had central lacteal expansion and Chiu scores of 2. Two rats had no obvious lesions and Chiu scores of 0. The Chiu score is showed in Fig. 1. Intestinal mucosal damage was evident in groups C and B2. The damage in group B2 was less than that in group C (Table 1). There were statistically significant differences in the intestinal villi length and mucosal thickness when groups B1, B2 and C were compared with group A (P < 0.05; Table 2).
Serum citrulline concentrations The serum citrulline concentrations of groups B1, B2 and C were lower than that of group A (P < 0.05). Serum citrulline concentrations of subgroups B1 and B2 were lower than that of group C (P < 0.05; Table 3).
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Chiu scores.
Group Group Group Group Group
A B1 B2 C
Number of cases
Chiu score
10 5 5 10
0.1 0.2 1.4 1.0
± ± ± ±
0.416 0.447 1.140a,b 0.667a
Note: F = 6.831, P = 0.002. a P < 0.05 versus group A. b P < 0.05 versus group C.
Serum I-FABP concentrations Serum I-FABP concentrations of groups B1, B2 and C were higher than in group A (P < 0.05); I-FABP concentrations of subgroups B1and B2 were higher than in group C (P < 0.05). The I-FABP concentrations in subgroups B1and B2 also were significantly different (Table 4).
Figure 2 Correlation of serum citrulline and I-FABP concentrations in Group C. Data represent mean ± SD, P < 0.01, R = —0.844 for Pearson correlation analysis.
Intestinal glutamine concentrations Intestinal glutamine concentrations in subgroups B1 and B2 were lower than those in groups A and C (P < 0.05). The concentrations in subgroups B1and B2 were also significantly different (Table 5). Correlation analyses In group C, the serum citrulline concentration was negatively correlated with I-FABP concentration (P < 0.01, R = —0.844), (Fig. 2). In group C, the serum citrulline concentration was negatively correlated with intestinal Chiu score (P < 0.01, R = —0.850), (Fig. 3).
Discussion Currently, the intestinal tract is considered the key to MODS, the amplifier of inflammation, and origin of systemic bacteremia and toxemia [1]. However, due to the body’s protective mechanisms, the intestinal tract is also the most easily-damaged organ [22]. In states of low perfusion, the body redistributes systemic blood flow to protect organs, such as the heart and brain, by reducing the blood flow to the limbs and intestinal tract [22]. When intestinal blood perfusion is reduced, gastrointestinal tissue oxygen supply also decreases, leading to potential intestinal tract damage, and ultimately gastrointestinal dysfunction/failure (GIDF) [22].
Table 2 Group Group Group Group Group
A B1 B2 C
Figure 3 Correlation of serum citrulline concentrations with intestinal Chiu scores in Group C. Data represent mean ± SD (P < 0.01, R = —0.850).
GIDF is common in critically ill patients [23]. There is at least one gastrointestinal symptom in nearly 59% of all intensive care unit (ICU) patients, and there is an association with increased mortality [24]. The ability to sustain or improve nutritional status, and maintain intestinal mucosal barrier function, depends on adequate function of intestinal epithelial cells [1]. Sepsis
Villus measurements. Number of cases
Villus length (m)
10 5 5 10
284.628 239.816 260.060 259.583
± ± ± ±
15.4321 15.3801a 30.508a 17.2101a
Mucosal thickness (m) 539.551 430.450 455.820 449.319
± ± ± ±
44.936 39.0591a 50.956a 33.7501a
Villi length:mucosal thickness 0.528 0.557 0.571 0.578
Note: villi length, F = 6.800, P = 0.002; mucosal thickness: F = 11.551, P = 0.001. a P < 0.05 versus group A.
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Serum citrulline levels in SAID Table 3 Group Group Group Group Group
A B1 B2 C
5
Serum citrulline concentrations (mol/L) (¯ x ± s). Number of cases
Serum citrulline (umol/L)
10 5 5 10
72.568 39.039 38.072 66.997
± ± ± ±
4.161 2.213a,b 1.980a,b 6.1751
Note: Blood was collected 12 hours after the final feedings (F = 107.995, P < 0.001). a P < 0.05 versus group A. b P < 0.05 versus group C.
Table 4
Serum I-FABP Concentrations (ng/L) (¯ x ± s).
Group Group Group Group Group
A B1 B2 C
Number of cases
Serum I- FABP (ng/L)
10 5 5 10
1.228 4.990 10.841 3.044
± ± ± ±
0.123 0.417a,b 1.254a,b,c 0.2961
Note: Blood was collected 12 hours after the final feedings (F = 22.295, P < 0.001). a P < 0.05 versus group A. b P < 0.05 versus group C. c P < 0.05 versus group B1.
is caused by infection of systemic inflammatory response syndrome (SIRS). During sepsis, intestinal epithelial cell apoptosis increases, and the number of cells decreases resulting in epithelial cell dysfunction, damage to intestinal mucosa, intestinal flora imbalance, and inhibition of intestinal immune function. This leads to endotoxin release from the lumen into the blood, and causes increased release of inflammatory mediators, further damaging the intestinal mucosa. Translocation of a large number of bacteria and endotoxins causes the body to increase inflammatory mediators, leading to a vicious cycle resulting in MODS [1,25]. Citrulline is involved in the nitric oxide (NO) cycle, including local recycling of citrulline [26]. It is also involved in the urea cycle in the liver [26]. Finally, citrulline affects the intestinal-renal axis wherein glutamine is converted to arginine [3]. Citrulline is mainly generated through the absorption and metabolism of glutamine by epithelial cells of the small intestine. Although other amino acids such as arginine, proline and ornithine are also involved in the generation of citrulline [13], an isotope-based study showed
Table 5 Glutamine concentrations in intestinal tissue (ng/L) (¯ x ± s). Group Group Group Group Group
A B1 B2 C
Number of cases
Intestinal glutamine (ng/L)
10 5 5 10
2.924 1.023 1.651 2.320
± ± ± ±
0.135 0.039a,b 0.036a,b 0.157
Note: Twelve hours after the final feedings, rats were sacrificed for pathological examination of the ileum. F = 302.220, P < 0.001. a P < 0.05 versus group A. b P < 0.05 versus group C.
that, in humans, 80% of citrulline is generated from the metabolism of glutamine [27]. The glutamine in the intestinal tract or arterial blood is converted into citrulline through the glutamate-ornithine pathway of epithelial cells in the small intestine [28,29]. Citrulline is then released into the hepatic portal vein system. In the current study, we found that citrulline levels in the sepsis model group were significantly lower than those of the control group. Our data are in line with those of Crenn et al. [11] who reported a significant decrease in serum citrulline levels when digestive bacterial translocation occurred at the onset of septic shock. The authors stated that the decreased citrulline levels could be a reflection of early acute intestinal dysfunction. Many recent findings have shown that citrulline can reflect acute decreases in the number of intestinal epithelial cells, and can reflect intestinal toxicity of radiation and chemotherapy [7,30]. It can also serve as an early indicator of acute rejection reaction after intestinal transplantation [28]. Following high-dose chemotherapy, decreases in serum citrulline concentrations are associated with bacteremia [31]. Pediatric studies have also confirmed a correlation between the reduction of serum citrulline concentration and mucosal damage [32]. The current data indicate that the I-FABP concentrations were significantly higher in the sepsis model group, leading to a negative correlation with citrulline in group C (P < 0.01, R = —0.844). It was also found that serum citrulline concentration was negatively correlated with intestinal Chiu score (P < 0.01, R = —0.850). A recent study showed that citrulline level could be used as an indicator of intestinal failure in patients with acute pancreatitis [33]. This study found that the serum citrulline concentrations in patients with severe pancreatitis decreased significantly compared to those with mild pancreatitis, and exhibited a negative correlation with IFABP. The I-FABP was clearly correlated with the extent of gastrointestinal disorder, acute physiology and chronic health evaluation II scores (APACHE II), C-reactive protein levels and ICU stay durations. This study confirmed that, in patients with SIRS and hypovolemia-induced clinical severe intestinal disorders, the degree of intestinal dysfunction was associated with the intestinal epithelial cell necrosis, which was reflected by I-FABP. A decrease in serum citrulline concentrations confirmed the reduction of intestinal epithelial cells. However, in the current study, there was no correlation between citrulline and I-FABP in groups B1 and B2. This inconsistency might have been related to the small sample size, and time points selected. Epithelial cells of the small intestine are the main sources of serum citrulline. A reduction of intestinal epithelial cells during ischemia would cause a decrease in serum citrulline levels. During SIRS, dysfunction of intestinal epithelial cells would also lead to a decline in serum citrulline levels. Hietbrink et al. [34] found that SIRS increased intestinal permeability, while the level of I-FABP did not change in endotoxemia patients. This indicates that SIRS caused increases in permeability, but did not directly damage intestinal epithelial cells. Therefore, the number of intestinal epithelial cells did not decrease at that time. There was only intestinal dysfunction, and yet serum citrulline levels still declined. Because the synthesis of intestinal citrulline occurs in the mitochondria of intestinal epithelial cells, the SIRS-induced mitochondrial dysfunction might lead to the
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decline of serum citrulline levels. In the current study, the 36-h subgroup was created in consideration of this situation. The citrulline concentrations decreased at 36 h, while the I-FABP concentrations significantly increased. This inconsistency might have been related to the small sample size, and time points selected. The current study used the citrulline generation test (CGT) [13]. The citrulline metabolic pathway revealed that the above studies could not completely exclude a lack of glutamine, a citrulline metabolic precursor. Glutamine is an essential nutrient for maintaining intestinal mucosal metabolism, structure and function when patients are under stress. There have been some reports that the glutamine concentrations of muscles and plasma in critically ill patients are reduced [12]. This has led to studies of the CGT [13]. The CGT is based on the assumption that functional competent enterocytes are required to convert glutamine to citrulline. Thus, citrulline produced by the intestine from a fixed load of glutamine, through enteral or intravenous administration, could reflect the function of mucosal epithelial cells. Glutamine administered as an alanine-glutamine dipeptide was used so that the test results would not be affected by the amount of basic glutamine storage [1]. The current study used the common nutritional supplement (peptisorb). In group B, Group C received additional glutamine by gavage, and the glutamine concentration was significantly higher than in the Pepti-only group. Thus, the factor of insufficient substrate was excluded; citrulline better reflected the number and function of intestinal epithelial cells. The hepatic circulatory system has no first-pass effect on citrulline. Liver-produced citrulline can be taken up by the kidney and metabolized to arginine [27], and therefore, citrulline levels can be affected by renal function. This could limit the application of citrulline in assessing the functions of small intestinal epithelial cells, because most critically ill patients have various degrees of renal impairment. The current study did not consider the impact of renal function. This will be considered in a future study. However, some studies have found that even if renal failure was present, if the fasting serum citrulline concentration was below the reference value (< 20 mol/L), it could still reflect the function of intestinal epithelial cells [35]. We conclude that serum citrulline levels could reflect the number and function of intestinal epithelial cells, and might be useful as a non-invasive indicator in the diagnosis of acute intestinal dysfunction in critically ill patients. The feasibility of serum citrulline as a common indicator of clinical detection still needs to be confirmed by human studies.
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Disclosure of interest [16]
The authors have not supplied their declaration of conflict of interest.
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