Effects of glutamine on wound healing

Abstract

Studies reporting the need for replacing amino acids such as glutamine (Gln), hydroxymethyl butyrate (HMB) and arginine (Arg) to accelerate wound healing are available in the literature. The primary objective of this study was to present the effects of Gln on tissue hydroxyproline (OHP) levels in wound healing. This study was conducted on 30 female Sprague Dawley rats with a mean weight of 230 ± 20 g. Secondary wounds were formed by excising 2 × 1 cm skin subcutaneous tissue on the back of the rats. The rats were divided into three equal groups. Group C (Control): the group received 1 ml/day isotonic solution by gastric gavage after secondary wound was formed. Group A (Abound): the group received 0·3 g/kg/day/ml Gln, 0·052 g/kg/day/ml HMB and 0·3 g/kg/day/ml Arg by gastric gavage after secondary wound was formed. Group R (Resource): the group received 0·3 g/kg/day/ml Gln by gastric gavage after secondary wound was formed. The OHP levels of the tissues obtained from the upper half region on the 8th day and the lower half region on the 21st day from the same rats in the groups were examined. Statistical analysis was performed using the statistics program SPSS version 17.0. No statistically significant differences were reported with regard to the OHP measurements on the 8th and 21st days (8th day: F = 0·068, P = 0·935 > 0·05; 21st day: F = 0·018, P = 0·983 > 0·05). The increase in mean OHP levels on the 8th and 21st days within each group was found to be statistically significant (F = 1146·34, P = 0·000 < 0·001). We conclude that in adults who eat healthy food, who do not have any factor that can affect wound healing negatively and who do not have large tissue loss at critical level, Gln, Arg and HMB support would not be required to accelerate secondary wound healing.

Introduction

Normal wound healing consists of four phases: haemostasis, inflammation, proliferative and remodelling. During the proliferative phase, tissue granulation, epithelisation and collagen production occur. An increase in fibroblasts occurs during the proliferative phase in normal wound healing (i.e. the fibrotic index increases). Collagen production and release begin the third day and continue for 3 weeks. Collagens release from fibroblasts and their cross‐linkage enhances wound tension strength. The amount and quality of collagen synthesis determine the wound tension strength, which is the mechanical integrity of the wound. The final phase of wound healing is the remodelling phase, which is characterised by the reorganisation of collagen fibrils and gradually increasing wound tension strength 1, 2. There are many studies available that report the need for replacing amino acids such as glutamine (Gln), hydroxymethyl butyrate (HMB) and arginine (Arg) to increase wound healing 1, 3, 4, 5, 6, 7. Gln is a free, non‐essential amino acid, which is found in substantial amount in the human body 8, 9. Gln deposits decrease more than 50% and plasma levels decrease more than 25% and continue to be low for a long time, following catabolic events such as sepsis 10. The Arg is a semi‐essential amino acid received with diet or which emerge as a result of endogenous catabolic reaction of citruline 1. However, it becomes essential in critical patients and serious trauma 6, 7. HMB appears in the body as leucine metabolite 7.

Increase in the collagen amount in the wound area affects wound healing positively if there is no factor preventing the regular sequence of collagen fibres 11, 12. Hydroxyproline (OHP) is an amino acid found in significant amounts in collagen structure and is used for determining the collagen of the tissue 8. In this study, the tissue OHP level was studied in determining the amount of collagen in the wound area.

The primary objective of this study is to present the effects of Gln tissue on OHP level in wound healing. The secondary objective is to research whether concomitant use of Arg and HMB—well known to have positive effects on wound healing—with Gln shall contribute to tissue OHP level.

Methods

This study was conducted at the Karadeniz Technical University Surgical Research Center, using rats from Karadeniz Technical University Surgical Research Center from 1 to 30 June 2012 after approval by the Karadeniz Technical University Animal Care and Local Ethics Committee (Approval Number: 2). This study was conducted on 30 female Sprague Dawley rats with a mean weight of 230 ± 20 g (10–12 weeks). During the study, all rats were kept in metal cages—one rat per cage—12 hours in illuminated and 12 hours in dark environment at normal room temperature (21°C ± 2°C) and humidity (40–60%) and were fed with standard rat feed and tap water. Rat care in cage was performed regularly with daily controls. All rats in the study were treated humanely in accordance with ‘Guide for the Care and Use of Laboratory Animals’. All the surgical interventions on the rats were performed under anaesthesia. Ketamine hydrochloride (Ketalar^® vial, 50 mg/ml; Eczacibasi, Istanbul, Turkey), 10 mg/kg intraperitoneal (ip), and xylazine hydrochloride (Rompun^® vial, 23·32 mg/ml; Bayer, Istanbul, Turkey), 5 mg/kg ip, were used for anaesthesia induction. The back regions of the rats were shaved under anaesthesia and the secondary wound model was created by excising 2 × 1 cm large cutaneous and subcutaneous tissue after the surgery areas were disinfected with povidone iodine under operating room conditions. Five millilitres of subcutaneous fluid resuscitation was applied to rats right after the secondary wound was created and after the eighth day the tissue sample was taken.

Group C (Control): The group that received 1 ml/day isotonic solution by gastric gavage after the secondary wound was formed.

Group A (Abound): The group that received 0·3 g/kg/day/ml Gln, 0·052 g/kg/day/ml HMB and 0·3 g/kg/day/ml Arg (Abound; Abbott Laboratories, Istanbul, Turkey) by gastric gavage after the secondary wound was formed.

Group R (Resource): The group that received 0·3 g/kg/day/ml Gln (Resource Glutamine; Nestle, Istanbul, Turkey) by gastric gavage after the secondary wound was formed.

Starting from the sixth hour after the formation of secondary wound, rats in groups A and R received a single dose of 0·3 g/kg/day/ml Gln by gastric gavage and its replacement was performed at the same time. Rats in group A received 0·052 g/kg/day HMB and 0·3 g/kg/day/ml Arg together with Gln. Rats in group C received a single dose of 1 ml/day isotonic solution by gastric gavage in the same hour. For analgesia, 20 mg/kg dose paracetamol was added to daily drinking waters of the rats in all groups. Sedation was not performed on rats in gastric gavage administration. For Gln replacement, Gln amount contained in two different oral Gln preparations available in the market (Abound; Resource Glutamine) was used at an equal rate of Gln by diluting with 1 ml isotonic solution.

Daily wound dressing was performed on the rats. Tissue samples taken from the upper half part of the wound area on the 8th day and from the lower half part of the wound area on the 21st day from the same rats in all groups under anaesthesia were placed in eppendorf tubes. The weights of all the rats were measured and recorded at the beginning and on the 21st day. Tissue samples taken were stored at −80°C until tissue OHP levels were analysed. After tissue samples were taken, rats were euthanised under high‐dose anaesthesia with intracardiac puncture.

Biochemical method

Tissue OHP levels were determined by BioVision Hydroxyproline assay kit (Cat. No; K555‐100, Milpitas, CA) following the kit protocol and OHP levels were expressed as microgram per well.

Statistical method

During the evaluation of data obtained in the study, statistics program, SPSS 17.0 was used for the statistical analysis. While evaluating the study data, Shapiro–Wilk diffusion test as well as the descriptive statistical methods (mean, SD) were used for the evaluation of normal distribution (n < 30 and when outlier was not seen). Repeated measures analysis of variance (ANOVA) was used to determine intergroup (between) and intragroup (within) differences. Pairwise comparisons were evaluated with Bonferroni test. Results were evaluated with 95% confidence interval and P < 0·05 significance level and P < 0·001 advanced significance level.

The strength of this study was estimated with GPower 3.1 program. Because ten rats per group in three groups were included in the study, the strength was found to be (1 − β) 44·2% with 5% significance level and effect size 0·40 with 30 rats in total.

Results

No statistically significant differences within groups were reported with regard to the mean weight of rats (F = 0·003; P = 0·997 > 0·05). Also, no statistically significant differences were reported between the mean weights of rats in the beginning and on the 21st day (Bonferroni test; P = 0·304 > 0·05).

No statistically significant differences were determined between groups on the 8th and 21st days with regard to OHP measurements (8th day: F = 0·068, P = 0·935 > 0·05; 21st day: F = 0·018; P = 0·983 > 0·05). Increase in mean OHP levels on the 8th and 21st days within each group was found to be statistically significant (F = 1146·34, P = 0·000 < 0·001). Median OHP levels of rats in groups are presented in Table 1 and Figure 1.

Table 1.

Median hydroxyproline (OHP) levels

	Group C		Group A		Group R		F	P
	Median	SD	Median	SD	Median	SD
OHP (8th day)	1·41	0·25	1·42	0·24	1·40	0·22	0·068	0·935
OHP (21st day)	6·62	0·67	6·68	0·74	6·59	0·72	0·018	0·983
F			1146·34
P			0·000*

SD, standard deviation.

^* P < 0·001.

Figure 1.

Hydroxyproline levels.

Discussion

For a normal wound healing, sufficient protein intake is essential because collagen synthesis is inhibited when insufficient protein is taken 1. Amino acids such as Gln and Arg are required for optimal collagen synthesis. Biochemically, three principal amino acids in the structure of collagen are glycine, proline and OHP. A total of 99·8% of the total body OHP is found in the collagen molecule 8. Therefore, measurement of OHP level was used to determine the tissue collagen amount in this study.

It is known that Gln, Arg and HMB are critically important for wound healing. These molecules are either received as a diet or synthesised in the body. They also have an essential role in sepsis and critical intensive care patients 1, 7, 9, 10. Thus, for individuals who eat healthy food, deficiency of these molecules is not expected, with the exception of the serious factor like sepsis that can prevent the synthesis and intake of molecules with diet. Thus, adequate nutrition was found to be the best method for collagen synthesis in the study conducted by Barbul 8. However, daily protein intake at old ages should be higher because of the decrease in the ability of body for protein synthesis 1. Wound healing can be effected negatively in the presence of factors that increase collagen synthesis such as chronic malnutrition, diabetes mellitus, uraemia and advanced age 3, 13. Therefore, in such cases, to increase wound healing, the requirement for the replacement of molecules like Gln, Arg and HMB that increase collagen synthesis cannot be denied. Nevertheless, the essential question to be answered is whether additional molecules containing Gln, Arg and HMB are necessary to increase wound healing in young patients, eating properly and without any additional disease, because these molecules required for collagen synthesis shall be sufficient in the body. Because using preparations containing these molecules in patients eating healthy food to increase wound healing would bring additional treatment cost in clinical practice. In this study, we aimed to contribute to the resolution of this problem.

Besides the presence of studies reporting that Gln replacement increases tissue OHP levels 3, 4, 7 in the literature there are also studies reporting that it does not provide any contribution 14, 15, 16. In the study performed by Ozturk et al. 3 it was reported that preoperative enteral Gln replacement increased the tissue collagen amount in anastomosis line in rats to which colon anastomosis was performed. When it is considered that tissue OHP level decreased by 40% in the first 3 days in anastomosis line 17 and anastomosis strength increased with the trophic effect on mucosa of Gln as the primary energy source of enterocytes 18, in the study performed by Ozturk et al. 3 the result showing that Gln replacement in addition to normal diet increased tissue OHP level was an expected result. In our study, in contrast to this study, it was shown that Gln replacement did not improve tissue OHP level in the wound area. We consider that the differences in the results obtained are acceptable when experimental models used in both studies are taken into account. The studies conducted by McCauley et al. 14, 15 presented similar results to our study, stating that Gln addition to parenteral nutrition in rats to which colon anastomosis was performed did not make any contribution to wound healing. When the result of these studies is compared with the study conducted by Ozturk et al. 3, it can be commented that if there is sufficient Gln present in the body, additional Gln replacement is not required to increase wound healing, but it may have a contribution when administered enterally in colon anastomosis because of the topical trophic effect of Gln. In the study performed by da Costa et al. 4 it was reported that oral Gln replacement in preoperative and postoperative periods in rats to which colon anastomosis was performed increased collagen level in anastomosis line. And in the study conducted by Gokpinar et al. 16 it was shown that tissue OHP level increased significantly in colon anastomosis applied rats in groups where early enteral feeding was performed. However, in the same study, it was shown that the addition of Gln to enteral nutrition did not have a significant contribution to tissue OHP level. This result showed in accordance with our study that there is no need for additional Gln support to increase wound healing when sufficient nutritional support is provided.

It is seen that the studies found in literature, on the effects of Gln on tissue OHP levels, are performed mostly on colon anastomosis. These studies show that generally in colon anastomosis while replacement of Gln parenterally does not have any contribution on tissue OHP level, replacement of Gln enterally has an effect. These results showed that tissue OHP level does not increase with additional Gln support when sufficient amounts of Gln is present in the body, but that it increases depending upon the topical effect when performed enterally owing to specific trophic effect of Gln on colonic mucosa.

While there are many studies available in the literature regarding the effects of Gln on tissue OHP level in colon anastomosis, studies regarding its effects on secondary dermal wounds caused by burns and tissue loss are considerably limited. The study conducted on mice by Jalilimanesh et al. 5 stated that in contrast to our study, Gln replacement in the second‐degree burns has a positive effect on wound healing. Considering that the model used in this study was burn and the degree of burn was serious, requirement for additional Gln support was inevitable. In this study, while approximately 1·75 cm² burns are created in mice, in our study secondary wounds are created in order to develop 2 cm² cutaneous and subcutaneous tissue loss in rats.

The presence of Arg, another amino acid that has a role in collagen synthesis, in the body in sufficient amounts is essential for wound healing 1. In the study conducted by Seifter et al. 6, determination of decrease in tissue OHP levels in rats fed with low Arg diet in perioperative period shows the requirement for sufficient amounts of Arg for wound healing. It is also reported that HMB regulates protein turnover, decreases muscle breakdown and when combined with exercise increases muscle mass 7. However, the number of studies showing the effects of HMB on tissue OHP level in wound area is very limited 7. The study conducted by Williams et al. 7 reported that daily Gln, Arg and HMB administration to patients with deltoid implants increases collagen level. Unlike this study, in our study no significant increase in tissue collagen levels was detected in the groups that received Gln only, Arg with Gln and HMB with Gln in early and late periods. It is seen that in both studies doses of these three molecules are close to each other. We believe that the difference between these two studies lies on the fact that the study conducted by Williams et al. 7 is performed on patients with ages 70 and above. It is a well‐known fact that protein level to be taken with the diet should be higher in older patients because the ability of the body to synthesise protein and collagen decreases with increased age 1, 3. In line with our research, we did not come across any other study, apart from the one conducted by Williams et al., showing the difference on tissue OHP levels caused by the concomitant use of these three molecules 7.

In conclusion, there should be sufficient amounts of Gln, Arg and HMB in the body for healthy wound healing. All these molecules are taken as part of a diet or are synthesised in the body. While wound healing is effected negatively in the absence of these molecules, there is not sufficient evidence to show that presence of excessive amounts of these molecules accelerates wound healing. In accordance with the data obtained from this study, we consider that there is no need for additional Gln, Arg and HMB support to accelerate wound healing in secondary wounds in adults who eat healthy food and who do not have a factor that can affect wound healing negatively and a critically large tissue loss. Thus, a careful assessment performed for deciding the patients to whom replacement shall be performed may decrease treatment costs significantly.