Parenteral glutamine supplementation in acute inflammatory conditions is controversial. We evaluated the inflammatory and survival responses after parenteral glutamine infusion in sodium taurocholate-induced acute pancreatitis (AP) model. Lewis rats received 1 g/kg parenteral glutamine (
Glutamine can become essential during hypercatabolic stress and under critical conditions, such as severe trauma, sepsis, inflammatory diseases, and burns [
In critically ill patients, glutamine supplementation has been suggested to properly support increased cell proliferation rates, gut barrier protection, and inflammatory dysfunction attenuation [
These observations have challenged the development of new guidelines for safe glutamine supplementation and have made apparent the need for new experimental studies to better understand this nutrient’s mechanisms of action in critical illness. Experimental acute pancreatitis (AP) is an effective model for the study of systemic responses that can be applied to test immunomodulatory therapies [
Adult male isogenic Lewis rats (
Animals were anesthetized with an intraperitoneal injection of ketamine (Ketamin-S(+)®, 100 mg/kg body weight; Cristália, Itapira, Brazil) and xylazine (Rompum®, 8 mg/kg body weight; Bayer, São Paulo, Brazil). Intravenous access was achieved by jugular central venous catheterization (CVC), according to a standard technique, followed by connection to a swivel apparatus that allowed the animals to have free mobility [
After 72 h intravenous access, all animals were anesthetized with an intraperitoneal injection of 100 mg/kg body weight ketamine (Ketamin-S(+)®, Cristália) and 8 mg/kg body weight xylazine (Rompum®, Bayer). The pancreas was exteriorized through an abdominal incision and the pancreatic duct was catheterized using a 24-gauge angicatheter. AP was then induced by retrograde injection of 0.5 mL 3% sodium taurocholate solution (Sigma Chemical, St Louis, MO, USA), according to a standard technique [
Blood samples were centrifuged at 1,000 ×g at 4°C for 10 min to obtain serum. Concentrations of cytokines (interleukin- [IL-] 1, IL-2, IL-4, IL-6, IL-10, interferon- [IFN-]
Approximately 100 mg lung tissue and 50 mg liver tissue were pulverized in liquid nitrogen. The material was homogenized in RIPA lysis buffer (100 mM Tris-HCl [pH 7.5], 1% sodium deoxycholate, 1% NP40, 150 mM NaCl, 0.1% sodium dodecyl sulfate [SDS]) plus protease inhibitors (1 mg/mL pepstatin A, 100 mM phenylmethylsulfonyl fluoride). The samples were then centrifuged at 14,000 ×g for 10 min at 4°C. The supernatants were collected and protein concentrations were quantified using the Bradford method (Bio-Rad Laboratories, Hercules, CA, USA).
Protein samples were added to sample buffer (2% SDS, 60 mM Tris [pH 6.8], 5% mercaptoethanol, 0.01% bromophenol blue) and subjected to electrophoresis in a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) system (1.5 M Tris-HCl, 10% SDS, 30% bis-acrylamide, 10% ammonia persulfate, and 3,3,5,5-tetramethylethylenediamine). Proteins were then transferred to nitrocellulose membranes using a semidry transfer apparatus (both from Bio-Rad Laboratories). The membranes were incubated in a blocking solution of 5% skim milk in TBST buffer (50 mM Tris buffer [pH 8.0], 100 mM NaCl, and 1% Tween 20) for 1 h at room temperature. Then, they were washed in TBST and incubated with the primary antibody against the protein of interest (HSP polyclonal goat anti-rat®; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C. Subsequently, the membranes were incubated in a solution containing the peroxidase-conjugated secondary antibody (1 : 1000; Santa Cruz Biotechnology), and Super Signal detection (Pierce, Rockford, IL, USA) was performed. Protein expression was compared by gel densitometry using the ImageJ public domain software created by Wayne Rasband at the US National Institutes of Mental Health, which has been used previously for the determination of HSP70 and HSP90 [
After AP induction, 20 rats in each group remained under observation for a maximum of 7 days, with access to standard oral diet (AIN-93M) and water ad libitum. The animals were observed individually every 8 h for death registration. Animals that survived until 7 days after AP were sacrificed with an intraperitoneal injection of 80 mg/kg ketamine hydrochloride (Ketamin-S(+)®; Cristália) and 8.0 mg/kg xylazine hydrochloride (Rompum® 2%; Bayer).
All inflammatory variables were compared using the Kruskal–Wallis and Behrens–Fisher tests, as Kolmogorov–Smirnov tests showed that they were not distributed normally. These comparisons were performed between groups at each time point and within groups over time. Mortality and survival data were evaluated by the Fisher test and Kaplan–Meier analysis, respectively. All analyses were based on a 5% level of significance and were performed using SPSS software (ver. 18.0 for Windows; SPSS, Chicago, IL, USA).
Serum cytokine levels did not differ among groups at any time point (Table
Serum cytokine levels in rats with acute pancreatitis, according to treatment and time point.
Cytokine | Treatment | Time point | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
2 h | 12 h | 24 h | |||||||||||
Median | 1° quartile | 3° quartile |
|
Median | 1° quartile | 3° quartile |
|
Median | 1° quartile | 3° quartile |
| ||
IL-1a | Saline | 14.41 | 4.67 | 47.89 | 0.552 | 15.60 | 11.40 | 31.86 | 0.574 | 1.84 | 1.84 | 46.08 | 0.583 |
Glutamine | 4.67 | 4.67 | 12.27 | 13.57 | 9.48 | 33.94 | 12.49 | 1.84 | 25.98 | ||||
Nontreatment | 8.47 | 4.67 | 47.89 | 8.57 | 6.13 | 17.86 | 72.61 | 1.84 | 128.00 | ||||
|
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IL-4 | Saline | 3.98 | 3.98 | 8.90 | 0.970 | 5.91 | 4.88 | 9.23 | 0.464 | 1.30 | 1.30 | 13.10 | 0.398 |
Glutamine | 3.98 | 3.98 | 3.98 | 5.91 | 3.91 | 15.38 | 1.30 | 1.30 | 4.88 | ||||
Nontreatment | 3.98 | 3.98 | 3.98 | 2.17 | 1.35 | 6.98 | 19.72 | 1.30 | 42.82 | ||||
|
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IL-2 | Saline | 25.50 | 22.32 | 31.21 | 0.833 | 49.40 | 10.97 | 72.34 | 0.814 | 74.03 | 27.12 | 117.21 | 0.249 |
Glutamine | 23.89 | 22.31 | 30.38 | 46.75 | 18.16 | 90.19 | 70.94 | 17.49 | 167.75 | ||||
Non-treatment | 23.10 | 17.69 | 33.71 | 37.89 | 24.44 | 48.50 | 135.50 | 83.31 | 166.00 | ||||
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IL-6 | Saline | 200.00 | 76.71 | 289.50 | 0.644 | 220.00 | 50.13 | 12704.00 | 0.971 | 369.00 | 184.21 | 766.50 | 0.458 |
Glutamine | 178.00 | 33.11 | 452.00 | 331.25 | 29.70 | 1619.00 | 903.00 | 486.50 | 1089.00 | ||||
Nontreatment | 336.00 | 33.11 | 3135.00 | 1789.00 | 113.00 | 5319.00 | 464.50 | 293.00 | 599.00 | ||||
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IL-10 | Saline | 400.00 | 294.50 | 560.00 | 0.288 | 142.00 | 99.08 | 691.00 | 0.964 | 65.83 | 34.62 | 183.18 | 0.553 |
Glutamine | 539.00 | 298.00 | 893.00 | 482.50 | 65.28 | 1521.00 | 77.82 | 54.16 | 125.41 | ||||
Nontreatment | 336.50 | 168.00 | 482.00 | 273.00 | 96.09 | 617.00 | 51.42 | 39.50 | 57.66 | ||||
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IFN- |
Saline | 72.62 | 30.44 | 89.41 | 0.142 | 6.50 | 6.50 | 6.50 | 0.908 | 4.01 | 4.01 | 4.01 | 0.776 |
Glutamine | 55.67 | 55.67 | 64.15 | 6.50 | 6.50 | 6.50 | 4.01 | 4.01 | 4.01 | ||||
Nontreatment | 85.25 | 64.15 | 93.61 | 6.50 | 6.50 | 6.50 | 4.01 | 4.01 | 63.08 | ||||
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TNF- |
Saline | 8.44 | 1.82 | 15.97 | 0.390 | 4.84 | 2.17 | 5.86 | 0.862 | 13.02 | 9.19 | 23.27 | 0.579 |
Glutamine | 11.22 | 1.82 | 11.22 | 3.91 | 2.17 | 8.05 | 16.31 | 3.94 | 18.50 | ||||
Nontreatment | 1.82 | 1.82 | 8.53 | 4.19 | 2.43 | 9.21 | 22.27 | 14.12 | 38.39 |
IL, interleukin; INF, interferon; TNF, tumor necrosis factor.
Median serum concentrations of interferon- (IFN-) gamma (a), interleukin- (IL-) 2 (b), IL-10 (c), and tumor necrosis factor- (TNF-) alpha (d) at 2, 12, and 24 h after acute pancreatitis induction with sodium taurocholate in Lewis rats previously treated or not treated with 48 h infusion of parenteral saline or glutamine.
Data on HSP expression are presented in Table
Heat shock protein expression in rats with acute pancreatitis, according to treatment and time point.
Variable | Treatment | Time point | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
2 h | 12 h | 24 h | |||||||||||
Median | 1° quartile | 3° quartile |
|
Median | 1° quartile | 3° quartile |
|
Median | 1° quartile | 3° quartile |
| ||
HSP70 lung | Saline | 15615447 | 14285740 | 20267619 | 0.372 | 7848 | 6817 | 9901 | 0.066 | 24064 | 21086 | 27654 |
|
Glutamine | 13645497 | 8114841 | 15673669 | 11684 | 11021 | 15036 | 23499 | 22976 | 26745 | ||||
Nontreatment | 12956058 | 10096912 | 16820669 | 7219 | 5854 | 10299 | 33718 | 28039 | 37863 | ||||
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HSP90 lung | Saline | 16654811 | 15125912 | 24525217 |
|
8079 | 6392 | 9511 | 0.065 | 14031 | 11409 | 28005 | 0.1067 |
Glutamine | 18875083 | 16923497 | 25849518 | 11742 | 9796 | 13052 | 8108 | 6184 | 9750 | ||||
Nontreatment | 8037912 | 7573083 | 12943462 | 8745 | 5670 | 10700 | 9094 | 7174 | 12214 | ||||
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HSP70 liver | Saline | 27154796 | 22116953 | 32188418 | 0.175 | 7497 | 6614 | 9658 |
|
24495 | 20696 | 24938 | 0.1495 |
Glutamine | 28513246 | 26185806 | 35924403 | 23252 | 15236 | 26715 | 15817 | 13442 | 20652 | ||||
Nontreatment | 34420660 | 30578261 | 41040332 | 9875 | 7099 | 11308 | 20686 | 14079 | 21387 | ||||
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HSP90 liver | Saline | NS | NS | NS | NS | 10014 | 7396 | 10263 |
|
19507 | 17368 | 25291 |
|
Glutamine | NS | NS | NS | 19218 | 15843 | 23452 | 16841 | 16074 | 21436 | ||||
Nontreatment | NS | NS | NS | 8671 | 5527 | 11237 | 337601 | 30857 | 37946 |
NS, nonsignificant expression to be detected; HSP, heat shock protein.
Lung and liver expression of heat shock proteins (HSPs) at different time points after acute pancreatitis induction with sodium taurocholate in Lewis rats previously treated or not treated with 48 h infusion of parenteral saline or glutamine. (a) Expression of HSP90 in lung tissue at 2 h. (b) Expression of HSP90 in liver tissue at 12 h. (c) Expression of HSP70 in liver tissue at 12 h. (d) Expression of HSP70 in lung tissue at 12 h. (e) Expression of HSP70 in lung tissue at 24 h. (f) Expression of HSP90 in liver at 24 h. Data are expressed as medians.
No significant difference in 7-day mortality was observed among groups (nontreatment, 28%; saline, 47%; glutamine, 27%) (Figure
Seven-day mortality rates of Lewis rats treated or not treated with 48 h parenteral infusion of saline or glutamine before acute pancreatitis induction with sodium taurocholate.
Our study aimed to contribute to the understanding of potential inflammatory mechanisms that may impact the risk-benefit balance of parenteral glutamine infusion in critical care. AP was chosen as a critical condition model due to the central roles of inflammatory mediators in its physiopathology and in multiple organ dysfunction syndrome, which is usually its primary cause of death [
In human and experimental models, marked release of the proinflammatory mediators IL-1, IL-6, and TNF-
The glutamine group also did not show the significant decreases in IFN-
Accordingly, immunotherapy with IFN-
In our study, marked effects on HSP expression were also observed in the glutamine group in relation to the other groups. This effect included increased liver HSP70 expression and a tendency for increased lung HSP70 expression 12 h after AP, as well as an early significant increase in lung and liver HSP90 expression. Xue et al. [
Increases in HSP70 expression induced by glutamine are associated with improvements in survival, tissue injury, and inflammatory response [
Parenteral glutamine supplementation was recently associated with high mortality rates in critically ill patients with multiple organ failure [
Our study has some limitations in addition to its experimental nature, which may limit the applicability of the findings to humans. First, glutamine was infused alone and before critical stress. As the release of inflammatory mediators is transient, this strategy was adopted to provide glutamine to cells and tissue in time to observe its modulatory effects on these mediators in our model. However, we do not know whether the same effect would be observed if glutamine were infused with other nutrients and in the presence of stress factors. In addition, parenteral supply of glutamine before critical stress cannot be applied fully in clinical practice. Second, saline was used as a parenteral control for glutamine. Saline hydration can attenuate AP by mitigating changes in pancreatic microcirculation and circulatory disorders of the intestinal wall, which facilitate bacterial translocation and perpetuate the inflammation mechanism [
Within these limitations, our data suggest that a high dose of parenteral glutamine protects against stress-induced organ damage by improving cytokine profiles and increasing HSP70 and HSP90 expression in our AP model. These protective effects are of particular interest for the treatment of critically ill patients. Further studies must seek to design a protocol for parenteral administration of glutamine that allows us to take clinical advantage of its potential benefits.
Dan L. Waitzberg has received speaking honoraria from Fresenius-Kabi, the manufacturer of parenteral glutamine solution.
This study was supported by the Fundação de Amparo a Pesquisa do Estado de São Paulo (Process no. 2011/02071-7). The authors thank Fresenius-Kabi for kindly providing the glutamine solution (Dipeptiven), Baxter Laboratory for lending the peristaltic pumps, Farmoterápica for partnership in preparing the parenteral solution, and João Ítalo Dias França for statistical discussion.