Abstract: Background. The effect of oral L-glutamine on wound healing in two groups of mice with superficial second-degree burns was evaluated. Methods. Thirty (30) male mice were included and randomly divided into two groups. First, the mice underwent general anesthesia, then an iron plate heated to 80˚C was placed on each animal’s skin for 1 second to create a second-degree burn injury. The test group mice received glutamine powder (1 g/kg/day) dissolved in water; the control group did not receive this supplementation. The variables were weight, burn wound surface area (cm3), wound contraction, and wound healing percentage on days 7, 10, 13, 16, 19, and 22. Serum levels of albumin, urea, and creatinine were assayed on days 1 and 22. Results. There was no significant difference between the groups regarding weight or serum albumin, urea, and creatinine levels. However, mean wound contraction was significant between the groups on days 7, 10, 13, 16, 19, and 22. Complete wound healing (100%) was achieved on day 22 in the test group compared to 71% healing in the control group. Conclusion. A significant and positive effect of oral glutamine on burn wound healing was found. However, further research is necessary in order to understand which stage of the healing process glutamine supplementation affected.   A burn injury is a significant problem because not only is the patient affected, but the social, economic, and psychological influences of a burn are distinctive from other diseases. The burn severity is dependent upon the size, depth, and the part of the body that has been affected.1 Natural wound healing follows a predictable pattern and can be divided into the following stages, which have some overlap: 1) homeostasis and inflammation; 2) proliferation; 3) maturation and remodeling. Glutamine is an important non-essential amino acid,2,3 and its intracellular concentration is higher than other amino acids.2 Skeletal muscles are the major source of glutamine synthesis and release; however, glutamine release is also found in both fat tissue and the lungs.2 Under normal conditions, glutamate and glutamine are the main fuels for intestinal enterocytes. The kidneys also use glutamine. The healing pattern can be changed via physiologic and pathologic alterations, but the liver is an exception in this regard. The range of glutamine supplementation tolerance in humans is 0.57 g–0.75 g daily.1,4 Glutamine supplementation is useful in reducing intestinal injuries and improving intestinal mucosa and systemic cell immunity.5,6   Several studies have evaluated the effect of glutamine and glutamate supplementation in enteral nutrition for hypercatabolic states (eg, burn injuries), and have shown that glutamate could be a substitute for glutamine.7 Some studies have shown that glutamine enhances the ability of neutrophils to destroy bacteria,8 while some studies failed to show any effects in wound healing.9,10 In burn injuries, reduction of plasma and muscular glutamine with muscular wasting, weight loss, and infection occur. Glutamine supplementation has been shown to decrease these events and reduce hospital stays for these patients.11,12   A Chinese burn institution studied the effects of oral glutamine on gastrointestinal metabolic parameters after severe burn injury.13 The results showed improvement in plasma glutamine levels and intestinal permeability with a decrease in plasma endotoxins, hospital stay, and costs. Significant improvement in wound healing was also observed in the group that received oral glutamine compared to the group that received a standard enteral diet regimen. In a study of severe burns using a mouse model, the effects of tube feeding with glutamine supplementation on immune cells of Peyer’s patches and IgA responses were analyzed. The results indicated that the intestinal immunity of the mice that received oral glutamine had improved.13   According to several studies, the immunity-stimulating effects of oral glutamine highlight its important role in the complex mechanisms of the immune system. Peng et al14 studied a group of patients who received standard diet with oral glutamine supplementation compared with a group that received only standard diet. After 10 days the levels of IL-2 had notably increased in the glutamine supplement group. The T cell count moderately rose in patients in the glutamine-supplemented group, which is an important finding, and led the authors to propose that oral glutamine stimulates local and systemic immunity.14 Almost 25% of gastrointestinal cells are immune cells whose effect on glutamine in infected patients or those who have a compromised immune system is important.15 Therefore, the aim of this trial was to determine the effect of oral L-glutamine administration on second-degree burn wound healing in NIH mice.

Materials and Methods

  Thirty (30) NIH mice with mean weight of 40 g–50 g were used for this study. Each animal was weighed and then administered general anesthesia through peritoneal injection of xylazine (0.8 cc), ketamine (2 cc), and distilled water (7.2 cc). Each animal’s back hair was shaved, and then an iron plate (heated to 80˚C) was placed on each animal’s skin for 1 second in order to create a 1.75 cm2 second-degree burn injury.16 Following burn creation, the burned area was covered with ChitoHeal Gel Spray (ChitoTech Co, Tehran, Iran) every other day. The gel has antibacterial, hemostatic, and pain relief properties. The study period lasted a total of 22 days. The day of burn creation was considered as day 1. Circadian period with 12 hours of darkness and 12 hours of light was implemented and the mice did not have any dietary restrictions.17 The mice were randomly divided into two groups: a test group that received 1 g/kg/day glutamine dissolved in water, and a control group that did not receive any medication. Both groups had access to water ad libitum. Blood biochemistry assays on days 1 and 22 were done by AutoAnalyzer RA 1000. Wound constriction and imaging were performed on days 7, 10, 13, 16, 19, and 22. The photos were taken with a digital camera. Complete wound healing and wound surface were determined according to a dermatologist’s clinical evaluation and photo observation through the Adobe Photoshop 7.0 software, respectively. Wound constriction was measured using the following formula: Wound constriction = wound surface on day 1 – wound surface on desired day 10   After extracting data, statistical analyses were done using repeated measurements test, Student’s t test, paired t test, and Fisher’s exact test. All the tests were performed using SPSS version 11 software (SPSS Inc., Chicago, IL). All values are reported as mean ± standard deviation; P < 0.05 was considered significant. All P values were two-tailed.

Results

  Three mice died during the study period. The data of 13 mice in the test group and 14 in the control group were analyzed. According to these results, the mean weights were not significantly different between groups from the beginning to the end of the study period (Table 1).   The biochemical parameters on day 1 and day 22 in both groups are shown in Table 2. The mean of any parameters was not significantly different between groups from the beginning to the end of the study.   The frequency distribution of complete wound healing on different days after initiation of the intervention in test and control groups is shown in Table 3. According to these results, on all days after intervention with the exception of day 7, complete wound healing was significantly higher in the test group compared to the control group. On day 7 there was no improvement in either group. However, in the test group, the complete wound healing percentage was 92.3% on day 19, and with the exception of one case, all mice achieved complete healing. In the control group, only 50% of the mice healed completely on day 19. On the last day (day 22), all test mice showed complete healing, while the healing rate for the control group was 71.4%.   The means of wound constriction on different days from the beginning to the end of the intervention are shown in Table 4. The means were significantly different on all days except on day 7. In fact, glutamine was effective regarding wound contraction and accelerated wound healing.

Discussion

  The results indicate that while glutamine did not have any significant effect on biochemical parameters, it did have a considerable effect on wound healing. The role of glutamine in burn wound healing has not been established completely, but there is a lot of evidence supporting the beneficial effects of glutamine for treating burns. Many studies have shown the positive effects of glutamine for improving cellular immunity, absorption, and intestinal mucosa function in burn injuries.15,18 The direct effects of glutamine on burn wound healing has yet to be reported in the literature. The most important finding of the present study was the significant and positive effect glutamine had on wound contraction in the test group mice. In previous studies, the effect of glutamine on immune system indices and intestinal function has been evaluated and showed a rise in immune cells that led to a decrease in infection rates.15,19,20 Glutamine also increased intestinal function that led to increased intestinal absorption and acceleration of normal or oral feeding that are among the main objectives of nutritional care.21 Another goal in nutritional care is to decrease the catabolism rate (prevention of muscular proteolysis and weight loss of more than 10%).22–24 In all the mentioned studies, with regard to improvement of other indices, the hospitalization duration decreased.   In the present study, mean weights were higher in both groups on day 22 compared to the first day, but this finding is not attributable to glutamine since the time variable was a significant factor. Mean albumin, urea, and creatinine in the two groups were not different on days 1 and 22, and glutamine did not have any effect on the measured biochemical variables. The authors predicted there would not be a change in serum albumin, urea, and creatinine levels since the induced wound was a superficial, second degree burn. Conversely, during the study period, the level of glutamine did not modify serum levels of albumin, urea, or creatinine. However, the change in mean weight in both groups was not from the effects of glutamine, but rather the length of the study period. The average wound contraction between test and control groups and on days 7, 10, 13, 16, 19, and 22 were significantly different. This finding is indicative of wound contraction and healing percentage. Mean wound contraction on day 7 in the test group was different from mean wound healing on other days. However, in the control group, a significant difference in mean wound healing began on day 13. Near the conclusion of the study, in both groups the significant difference in mean wound contraction decreased as a result of the time variable. This is mainly due to the fact that second degree burns usually take 2 to 3 weeks to heal.1,25   The present study did not specify which stage of wound healing glutamine affected. The study did convey the impact of glutamine on overall wound healing. It is likely that glutamine increased cellular immunity indices (eg, inflammatory/immunity cells such as T cells, which are involved in the initial stages of wound healing [homeostasis and inflammation]). In this stage, T cells are important when moving from the first stage to the second stage of healing (proliferation).3 Glutamine is the main source of energy for immune cells.26,27 Glutamine supplementation might improve immunity indices and have a role in the wound healing process when severe catabolism decreases serum glutamine levels, and its resources, due to a burn injury.

Conclusion

  Administration of glutamine supplements accelerated the wound healing process, and it seems that glutamine is effective in the initial stages of healing. Therefore, the authors recommend oral glutamine for patients with a burn injury. Further study is required to determine which stage of healing glutamine effects most significantly, and to elucidate the cellular and molecular effects of glutamine.

Acknowledgments

The authors thank Payame-Noor University for funding this project. Special thanks to Arezoo Flahatiyan and Mohsen Mirsmaeili who participated in data collection. The authors also appreciate the efforts the Mehr Laboratory Research and Clinical Center for Infertility personnel who helped with data collection, blood sampling, and lab tests. Dr. Jalilimanesh is from the Department of Plastic and Reconstructive Surgery, Savaneh & Sookhtegi Hospital, Yazd, Iran; Dr. Mozaffari-Khosravi is from the Department of Nutrition, Faculty of Health, Shahid Sadoughi University of Medical Sciences and Health Services, Yazd, Iran; Ms. Azhdari is the Drug and Food Deputy, Shahid Sadoughi University of Medical Sciences and Health Services, Yazd, Iran Address correspondence to: Hassan Mozaffari-Khosravi, PhD Shahid Sadoughi University of Medical Sciences Bahonar Square P.O. Box 734 Yazd, Iran Phone: +98 351 724 9333 Email: mozaffari.kh@gmail.com

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