The Effect of Amino Acids on Wound Healing: A Systematic Review and Meta-Analysis on Arginine and Glutamine

Under stress conditions, the metabolic demand for nutrients increases, which, if not met, may slow down or indeed stop the wound from healing, thus, becoming chronic wounds. This study aims to perform a systematic review and meta-analysis of the effect of arginine and glutamine supplementation on wound healing. PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines were followed for the systematic review and ten electronic databases were used. Five and 39 human studies met the inclusion criteria for arginine and glutamine, respectively. The overall meta-analysis demonstrated a significant effect of arginine supplementation on hydroxyproline content (MD: 4.49, 95% CI: 3.54, 4.45, p < 0.00001). Regarding glutamine supplementation, there was significant effect on nitrogen balance levels (MD: 0.39, 95% CI: 0.21, 0.58, p < 0.0001), IL-6 levels (MD: −5.78, 95% CI: −8.71, −2.86, p = 0.0001), TNFα levels (MD: −8.15, 95% CI: −9.34, −6.96, p < 0.00001), lactulose/mannitol (L/M) ratio (MD: −0.01, 95% CI: −0.02, −0.01, p < 0.00001), patient mortality (OR: 0.48, 95% CI: 0.32, 0.72, p = 0.0004), C-reactive protein (CRP) levels (MD: −1.10, 95% CI: −1.26, −0.93, p < 0.00001) and length of hospital stay (LOS) (MD: −2.65, 95% CI: −3.10, −2.21, p < 0.00001). Regarding T-cell lymphocytes, a slight decrease was observed, although it failed to reach significance (MD: −0.16, 95% CI: −0.33, 0.01, p = 0.07). Conclusion: The wound healing might be enhanced in one or at various stages by nutritional supplementation in the right dose.


Introduction
A wound is known as the disruption in the physical continuity of functional tissues [1][2][3][4]. The healing process begins immediately after an injury [5][6][7][8] and involves four phases [3,[9][10][11][12][13]. The healing process consists of a series of sequential and overlapping physiological phases or stages that can persist for years [9,[14][15][16][17], as shown in Figure 1. It is not a linear process and, depending on diverse extrinsic and intrinsic factors, such as growth factors and cytokines, it can progress both backward and forward through the stages.

Nutrition
Nutrition is recognized as a key factor in wound healing. Under conditions of stress such as trauma or after surgery, the nutritional demand is increased [18][19][20] in part due to cell proliferation and protein synthesis [21].
Arginine is a conditionally essential amino acid that is synthesized from citrulline in healthy humans [22] (Figure 2). Based on previous reviews, arginine has been shown to modulate the immune function, hormone secretion, and endothelial function as well as being a precursor to the synthesis of proline in animal and human trials [23][24][25].

Nutrition
Nutrition is recognized as a key factor in wound healing. Under conditions of stress such as trauma or after surgery, the nutritional demand is increased [18][19][20] in part due to cell proliferation and protein synthesis [21].
Arginine is a conditionally essential amino acid that is synthesized from citrulline in healthy humans [22] (Figure 2). Based on previous reviews, arginine has been shown to modulate the immune function, hormone secretion, and endothelial function as well as being a precursor to the synthesis of proline in animal and human trials [23][24][25]. Metabolism of L-Arginine to produce NO and metabolites involved in the wound healing process. Arginine can be catabolized via the NO synthase pathway. Here, L-Arginine can be converted to L-ornithine and urea by arginase I. Then, by the action of ornithine aminotransferase, ornithine is transformed into proline, which is needed for collagen synthesis. L-ornithine can also be converted to polyamines, which are required for cell proliferation by ornithine decarboxylase [22].
There are two pathways in wound healing involving arginine (a) the arginase pathway, which produces polyamines, as well as ornithine and proline. Polyamines are needed for cell proliferation, while the latter ones are required for the synthesis of colla-

Nutrition
Nutrition is recognized as a key factor in wound healing. Under conditions of stress such as trauma or after surgery, the nutritional demand is increased [18][19][20] in part due to cell proliferation and protein synthesis [21].
Arginine is a conditionally essential amino acid that is synthesized from citrulline in healthy humans [22] (Figure 2). Based on previous reviews, arginine has been shown to modulate the immune function, hormone secretion, and endothelial function as well as being a precursor to the synthesis of proline in animal and human trials [23][24][25]. Metabolism of L-Arginine to produce NO and metabolites involved in the wound healing process. Arginine can be catabolized via the NO synthase pathway. Here, L-Arginine can be converted to L-ornithine and urea by arginase I. Then, by the action of ornithine aminotransferase, ornithine is transformed into proline, which is needed for collagen synthesis. L-ornithine can also be converted to polyamines, which are required for cell proliferation by ornithine decarboxylase [22].
There are two pathways in wound healing involving arginine (a) the arginase pathway, which produces polyamines, as well as ornithine and proline. Polyamines are needed for cell proliferation, while the latter ones are required for the synthesis of colla- Figure 2. Metabolism of L-Arginine to produce NO and metabolites involved in the wound healing process. Arginine can be catabolized via the NO synthase pathway. Here, L-Arginine can be converted to L-ornithine and urea by arginase I. Then, by the action of ornithine aminotransferase, ornithine is transformed into proline, which is needed for collagen synthesis. L-ornithine can also be converted to polyamines, which are required for cell proliferation by ornithine decarboxylase [22].
There are two pathways in wound healing involving arginine (a) the arginase pathway, which produces polyamines, as well as ornithine and proline. Polyamines are needed for cell proliferation, while the latter ones are required for the synthesis of collagen; and (b) the inducible nitric oxide (NO) synthetase or iNOS pathway, which is a precursor of nitric oxide ( Figure 2). NO plays a key role in wound healing as it regulates cell proliferation, collagen formation, and wound contraction [22,26].
Glutamine is the most abundant amino acid found in human blood plasma. It is used as a source of energy for the cells to proliferate, including lymphocytes, macrophages, fibroblasts, and epithelial cells [21,23]. Similar to arginine, its concentration in plasma decreases under conditions of metabolic stress, such as injury, and its depletion is proportional to the acuteness of the trauma [27][28][29][30]. Metabolism of glutamine to arginine in human macrophages. Carbamoyl phosphate when combined with ornithine via OTC is converted to citrulline. Then citrulline is transformed into argininosuccinate and then into arginine by the action of ASS and ASL, respectively. Arginine can then be turned into nitric oxide or ornithine. Ornithine can be transformed into glutamine, and vice versa, via glutamate and pyrroline-5-carboxylate.

Study Selection
Inclusion criteria: studies selected were randomised controlled trials (RCTs) where patients above 18 years old, healthy or not, suffering from acute or chronic wounds were supplemented with arginine or glutamine ( Figure 4 and Figure 5).
Exclusion criteria: Studies that did not entail in vivo human studies involving supplementation with arginine or glutamine were excluded from the review. Studies involving participants below 18 years of age were excluded from the review due to the metabolic stress already occurring resulting from growth. Studies involving patients with diabetes, where data were not complete or the original data were not presented and studies in another language other than English, Spanish or French were excluded from the review.

Population
Adults above 18 years old, healthy or not, suffering from acute or chronic wounds.

Intervention
Diet supplemented with either arginine or glutamine for at least 5 days.

Comparator
A control group, either treated with a placebo or not treated.

Data Extraction and Management
Data for the meta-analysis were extracted from figures using WebPlotDigitizer [56], tables and the test from the articles, and the change in mean and standard deviation between the baseline and final values for each outcome were used for the meta-analysis. No publication date restrictions were applied. Units of measurements were converted to mg/dL for CRP as necessary. Additionally, median values were converted to means and 1st-3rd quartiles were transformed into standard deviations, respectively.

Quality Assessment
The risk of bias assessment was assessed by the Cochrane risk of bias tool [57]. The domains evaluated included the random sequence generation (selection bias), allocation concealment (selection bias), blinding of participants and personnel (performance bias), blinding of outcome assessment (detection bias), incomplete outcome data (attrition bias), selective reporting (reporting bias), and other bias. Low risk of bias is indicated by a plus (+), unclear risk of bias by a question mark (?), and high risk of bias by a minus (−).

Data Analysis
Changes from baseline for the intervention were compared with the control in all the parameters analyzed [57]. The pooling of the data was conducted with the metaanalytic methodology, utilizing Cochrane Review Manager 5.4.1 (2020) [58] for the different outcomes evaluated applying fixed effects, the mean differences (MDs) and odds ratio as a degree of effect extent. Nevertheless, for nitrogen balance and T-cell lymphocytes levels, data were converted into standardized mean difference (SMD) owing to the use of different measurement scales. Pooled effect size estimates are presented with their 95% confidence intervals (95% CI). When studies reported multiple results (i.e., multiple-dose), these were included in the meta-analysis as independent comparisons. Heterogeneity was assessed using I 2 and Chi 2 and considered significant when I 2 > 50%. Results were considered significant when the p-value was below 0.05.

Results
Five (5) and 39 studies on arginine and glutamine, respectively were included in the systematic review (Figures 4 and 5) (Tables 2 and 3).

Risk of Bias of Included Studies on Arginine
The risks of bias in the included studies are shown in Figure 6. 100% of the studies showed a low risk of bias in relation to the random sequence generation and allocation concealment. While less than 75% have demonstrated a low risk of bias with respect to blinding of participants and personnel. In terms of blinding of outcome assessment, incomplete outcome data and selective reporting, all the studies showed a low risk of bias except for Barbul et al. [59] which exhibited an unclear risk of bias. Regarding other risks of bias, less than 50% of the studies showed a low risk of bias whereas the Langkamp-Henken study [60] demonstrated a high risk of bias.
showed a low risk of bias in relation to the random sequence generation and allocation concealment. While less than 75% have demonstrated a low risk of bias with respect to blinding of participants and personnel. In terms of blinding of outcome assessment, incomplete outcome data and selective reporting, all the studies showed a low risk of bias except for Barbul et al. [59] which exhibited an unclear risk of bias. Regarding other risks of bias, less than 50% of the studies showed a low risk of bias whereas the Langkamp-Henken study [60] demonstrated a high risk of bias.

Risk of Bias of Included Studies on Glutamine
The risks of bias in the included studies are shown in Figure 7. Of the studies, 100% showed a low risk of bias in relation to the random sequence generation. All the studies have demonstrated a low or unclear risk of bias regarding blinding of participants except the Goeters et al. [61] and Xian-Li et al. [62] studies, which exhibited a high risk of bias. On the other hand, more than 50% of the studies showed an unclear and high risk of bias in terms of other biases. The high bias is the case of Engel et al. [63] and Goeters et al. studies [61]. With respect to the other risk of bias, more than 75% of the studies exhibited a low risk of bias regarding allocation concealment, blinding of outcome assessment, incomplete data and selective reporting.

Risk of Bias of Included Studies on Glutamine
The risks of bias in the included studies are shown in Figure 7. Of the studies, 100% showed a low risk of bias in relation to the random sequence generation. All the studies have demonstrated a low or unclear risk of bias regarding blinding of participants except the Goeters et al. [61] and Xian-Li et al. [62] studies, which exhibited a high risk of bias. On the other hand, more than 50% of the studies showed an unclear and high risk of bias in terms of other biases. The high bias is the case of Engel et al. [63] and Goeters et al. studies [61]. With respect to the other risk of bias, more than 75% of the studies exhibited a low risk of bias regarding allocation concealment, blinding of outcome assessment, incomplete data and selective reporting.

Effects of Interventions
Based on the systematic review and meta-analysis, one distinct area was identified under arginine: Collagen deposition (hydroxyproline content); and seven distinct areas were identified under glutamine: nitrogen balance; wound healing time; length of hospital stay and patient mortality; lactulose/mannitol ratio; C-reactive protein; cytokines (IL-6 levels, TNFα levels) and T-cell lymphocytes.

Arginine (Arg)
Research on the pharmacological effects of arginine supplementation has been mostly based on its use for acute wounds, although some trials have studied its effect on chronic wounds (Table 2).

Effects of Interventions
Based on the systematic review and meta-analysis, one distinct area was identified under arginine: Collagen deposition (hydroxyproline content); and seven distinct areas   Several studies have demonstrated that supplementation with arginine increases lagen deposition and, therefore, enhances wound-breaking strength. The wound-br ing strength is the force needed to disrupt a wound [67]. Barbul et al. [59] observed improvement in a randomized, controlled trial (RCT) in 36 healthy and non-smoking mans by supplementing their diet with 24.8 g of free arginine as arginine hydrochlo and 17 g of free arginine as arginine aspartate per day for 2 weeks. Hydroxyproline c tent was assessed as an index of the synthesis and deposition of new collagen in a p tetrafluoroethylene tube inserted in the wound site. An enhanced collagen depositio 137 and 74% was noted in the arginine hydrochloride (p = 0.028; 23.85 ± 2.16 nmol/cm) arginine aspartate (p = 0.028, 17.57 ± 2.16 nmol/cm) groups, respectively, following a nificant difference observed in the controlled group (p < 0.001; 10.1 ± 2.32 nmol/cm in c trols). These results were confirmed later by Nussbaum [64], who carried out a sim trial in 45 healthy elderly people, randomly supplemented or not with 17 g of arginine day for 14 days. An improvement was also observed in collagen synthesis through hydroxyproline deposition (17.4 ± 2 nmol/cm; p < 0.02) and T-cell-mediated immune fu tion.
Arginine also influences the nitrogen balance. Nevertheless, this balance impro ment has been reported in many, but not in all studies. In a randomized double-b controlled study by Debats et al. [65], specific parameters related to wound healing w measured after supplementing 30 g of arginine (n = 16) or placebo (n = 19) for 10 d Angiogenesis, assessed as the number of vessels per high power field significantly creased on day 10 (8.9 ± 3.1 in the arginine group vs. 8 ± 2.8 in the placebo group, p < 0.0 Citrulline, ornithine and NO levels, Several studies have demonstrated that supplementation with ar lagen deposition and, therefore, enhances wound-breaking strength ing strength is the force needed to disrupt a wound [67]. Barbul et a improvement in a randomized, controlled trial (RCT) in 36 healthy an mans by supplementing their diet with 24.8 g of free arginine as arg and 17 g of free arginine as arginine aspartate per day for 2 weeks. H tent was assessed as an index of the synthesis and deposition of new tetrafluoroethylene tube inserted in the wound site. An enhanced co 137 and 74% was noted in the arginine hydrochloride (p = 0.028; 23.85 arginine aspartate (p = 0.028, 17.57 ± 2.16 nmol/cm) groups, respectiv nificant difference observed in the controlled group (p < 0.001; 10.1 ± 2 trols). These results were confirmed later by Nussbaum [64], who c trial in 45 healthy elderly people, randomly supplemented or not with day for 14 days. An improvement was also observed in collagen sy hydroxyproline deposition (17.4 ± 2 nmol/cm; p < 0.02) and T-cell-med tion.
Arginine also influences the nitrogen balance. Nevertheless, th ment has been reported in many, but not in all studies. In a rando controlled study by Debats et al. [65], specific parameters related to w measured after supplementing 30 g of arginine (n = 16) or placebo Angiogenesis, assessed as the number of vessels per high power fi creased on day 10 (8.9 ± 3.1 in the arginine group vs. 8 ± 2.8 in the place Angiogenesis, chronic wounds (Table 2). Several studies have demonstrated that supplementation with arg lagen deposition and, therefore, enhances wound-breaking strength. ing strength is the force needed to disrupt a wound [67]. Barbul et a improvement in a randomized, controlled trial (RCT) in 36 healthy an mans by supplementing their diet with 24.8 g of free arginine as argi and 17 g of free arginine as arginine aspartate per day for 2 weeks. H tent was assessed as an index of the synthesis and deposition of new tetrafluoroethylene tube inserted in the wound site. An enhanced col 137 and 74% was noted in the arginine hydrochloride (p = 0.028; 23.85 ± arginine aspartate (p = 0.028, 17.57 ± 2.16 nmol/cm) groups, respectiv nificant difference observed in the controlled group (p < 0.001; 10.1 ± 2 trols). These results were confirmed later by Nussbaum [64], who c trial in 45 healthy elderly people, randomly supplemented or not with day for 14 days. An improvement was also observed in collagen sy hydroxyproline deposition (17.4 ± 2 nmol/cm; p < 0.02) and T-cell-med tion.
Arginine also influences the nitrogen balance. Nevertheless, thi ment has been reported in many, but not in all studies. In a random controlled study by Debats et al. [65], specific parameters related to w measured after supplementing 30 g of arginine (n = 16) or placebo ( Angiogenesis, assessed as the number of vessels per high power fie creased on day 10 (8.9 ± 3.1 in the arginine group vs. 8  Research on the pharmacological effects of arginine supplementation ha mostly based on its use for acute wounds, although some trials have studied its e chronic wounds (Table 2). Several studies have demonstrated that supplementation with arginine increa lagen deposition and, therefore, enhances wound-breaking strength. The wound ing strength is the force needed to disrupt a wound [67]. Barbul et al. [59] observ improvement in a randomized, controlled trial (RCT) in 36 healthy and non-smok mans by supplementing their diet with 24.8 g of free arginine as arginine hydroc and 17 g of free arginine as arginine aspartate per day for 2 weeks. Hydroxyproli tent was assessed as an index of the synthesis and deposition of new collagen in tetrafluoroethylene tube inserted in the wound site. An enhanced collagen depos 137 and 74% was noted in the arginine hydrochloride (p = 0.028; 23.85 ± 2.16 nmol/c arginine aspartate (p = 0.028, 17.57 ± 2.16 nmol/cm) groups, respectively, followin nificant difference observed in the controlled group (p < 0.001; 10.1 ± 2.32 nmol/cm trols). These results were confirmed later by Nussbaum [64], who carried out a trial in 45 healthy elderly people, randomly supplemented or not with 17 g of argin day for 14 days. An improvement was also observed in collagen synthesis throu hydroxyproline deposition (17.4 ± 2 nmol/cm; p < 0.02) and T-cell-mediated immun tion.
Arginine also influences the nitrogen balance. Nevertheless, this balance im ment has been reported in many, but not in all studies. In a randomized doubl controlled study by Debats et al. [65], specific parameters related to wound healin measured after supplementing 30 g of arginine (n = 16) or placebo (n = 19) for 1 Angiogenesis, assessed as the number of vessels per high power field significan creased on day 10 (8.9 ± 3.1 in the arginine group vs. 8 ± 2.8 in the placebo group, p < Lymphocyte proliferation,

Arginine (Arg)
Research on the pharmacological effects of arginine supplemen mostly based on its use for acute wounds, although some trials have stu chronic wounds (Table 2). Several studies have demonstrated that supplementation with argin lagen deposition and, therefore, enhances wound-breaking strength. T ing strength is the force needed to disrupt a wound [67]. Barbul et al. [ improvement in a randomized, controlled trial (RCT) in 36 healthy and mans by supplementing their diet with 24.8 g of free arginine as argini and 17 g of free arginine as arginine aspartate per day for 2 weeks. Hyd tent was assessed as an index of the synthesis and deposition of new co tetrafluoroethylene tube inserted in the wound site. An enhanced colla 137 and 74% was noted in the arginine hydrochloride (p = 0.028; 23.85 ± 2 arginine aspartate (p = 0.028, 17.57 ± 2.16 nmol/cm) groups, respectively nificant difference observed in the controlled group (p < 0.001; 10.1 ± 2.32 trols). These results were confirmed later by Nussbaum [64], who carr trial in 45 healthy elderly people, randomly supplemented or not with 17 day for 14 days. An improvement was also observed in collagen synth hydroxyproline deposition (17.4 ± 2 nmol/cm; p < 0.02) and T-cell-mediat tion.
Arginine also influences the nitrogen balance. Nevertheless, this ment has been reported in many, but not in all studies. In a randomi controlled study by Debats et al. [65], specific parameters related to wo measured after supplementing 30 g of arginine (n = 16) or placebo (n Angiogenesis, assessed as the number of vessels per high power field creased on day 10 (8.9 ± 3.1 in the arginine group vs. 8

Arginine (Arg)
Research on the pharmacological effects of arginine supplementation ha mostly based on its use for acute wounds, although some trials have studied its e chronic wounds (Table 2). Several studies have demonstrated that supplementation with arginine increa lagen deposition and, therefore, enhances wound-breaking strength. The wound ing strength is the force needed to disrupt a wound [67]. Barbul et al. [59] observ improvement in a randomized, controlled trial (RCT) in 36 healthy and non-smok mans by supplementing their diet with 24.8 g of free arginine as arginine hydroc and 17 g of free arginine as arginine aspartate per day for 2 weeks. Hydroxyproli tent was assessed as an index of the synthesis and deposition of new collagen in tetrafluoroethylene tube inserted in the wound site. An enhanced collagen depos 137 and 74% was noted in the arginine hydrochloride (p = 0.028; 23.85 ± 2.16 nmol/c arginine aspartate (p = 0.028, 17.57 ± 2.16 nmol/cm) groups, respectively, followin nificant difference observed in the controlled group (p < 0.001; 10.1 ± 2.32 nmol/cm trols). These results were confirmed later by Nussbaum [64], who carried out a trial in 45 healthy elderly people, randomly supplemented or not with 17 g of argin day for 14 days. An improvement was also observed in collagen synthesis throu hydroxyproline deposition (17.4 ± 2 nmol/cm; p < 0.02) and T-cell-mediated immun tion.
Arginine also influences the nitrogen balance. Nevertheless, this balance im ment has been reported in many, but not in all studies. In a randomized doubl controlled study by Debats et al. [65], specific parameters related to wound healin measured after supplementing 30 g of arginine (n = 16) or placebo (n = 19) for 1 Angiogenesis, assessed as the number of vessels per high power field significan creased on day 10 (8.9 ± 3.1 in the arginine group vs. 8 ± 2.8 in the placebo group, p < Lymphocyte proliferation, levels, TNFα levels) and T-cell lymphocytes.

Arginine (Arg)
Research on the pharmacological effects of arginine supplemen mostly based on its use for acute wounds, although some trials have stu chronic wounds (Table 2). Several studies have demonstrated that supplementation with argin lagen deposition and, therefore, enhances wound-breaking strength. T ing strength is the force needed to disrupt a wound [67]. Barbul et al. [ improvement in a randomized, controlled trial (RCT) in 36 healthy and mans by supplementing their diet with 24.8 g of free arginine as argini and 17 g of free arginine as arginine aspartate per day for 2 weeks. Hyd tent was assessed as an index of the synthesis and deposition of new co tetrafluoroethylene tube inserted in the wound site. An enhanced colla 137 and 74% was noted in the arginine hydrochloride (p = 0.028; 23.85 ± 2 arginine aspartate (p = 0.028, 17.57 ± 2.16 nmol/cm) groups, respectively nificant difference observed in the controlled group (p < 0.001; 10.1 ± 2.32 trols). These results were confirmed later by Nussbaum [64], who carr trial in 45 healthy elderly people, randomly supplemented or not with 17 day for 14 days. An improvement was also observed in collagen synth hydroxyproline deposition (17.4 ± 2 nmol/cm; p < 0.02) and T-cell-mediat tion.
Arginine also influences the nitrogen balance. Nevertheless, this b ment has been reported in many, but not in all studies. In a randomi controlled study by Debats et al. [65], specific parameters related to wou measured after supplementing 30 g of arginine (n = 16) or placebo (n = Angiogenesis, assessed as the number of vessels per high power field creased on day 10 (8.9 ± 3.1 in the arginine group vs. 8 ± 2.8 in the placebo NO, tal stay and patient mortality; lactulose/mannitol ratio; C-reactive protein; cyto levels, TNFα levels) and T-cell lymphocytes.

Arginine (Arg)
Research on the pharmacological effects of arginine supplementation mostly based on its use for acute wounds, although some trials have studied i chronic wounds (Table 2). Several studies have demonstrated that supplementation with arginine in lagen deposition and, therefore, enhances wound-breaking strength. The wo ing strength is the force needed to disrupt a wound [67]. Barbul et al. [59] ob improvement in a randomized, controlled trial (RCT) in 36 healthy and non-sm mans by supplementing their diet with 24.8 g of free arginine as arginine hyd and 17 g of free arginine as arginine aspartate per day for 2 weeks. Hydroxyp tent was assessed as an index of the synthesis and deposition of new collagen tetrafluoroethylene tube inserted in the wound site. An enhanced collagen de 137 and 74% was noted in the arginine hydrochloride (p = 0.028; 23.85 ± 2.16 nm arginine aspartate (p = 0.028, 17.57 ± 2.16 nmol/cm) groups, respectively, follo nificant difference observed in the controlled group (p < 0.001; 10.1 ± 2.32 nmol trols). These results were confirmed later by Nussbaum [64], who carried ou trial in 45 healthy elderly people, randomly supplemented or not with 17 g of a day for 14 days. An improvement was also observed in collagen synthesis t hydroxyproline deposition (17.4 ± 2 nmol/cm; p < 0.02) and T-cell-mediated im tion.
Arginine also influences the nitrogen balance. Nevertheless, this balanc ment has been reported in many, but not in all studies. In a randomized do controlled study by Debats et al. [65], specific parameters related to wound he measured after supplementing 30 g of arginine (n = 16) or placebo (n = 19) f Angiogenesis, assessed as the number of vessels per high power field signi creased on day 10 (8.9 ± 3.1 in the arginine group vs. 8 ± 2.8 in the placebo group IL-2 Arg: arginine; Cu: copper; Gln: glutamine; HBM: β-hydroxy-β-methylbutyrate; IGF-1: insulin-like growth factor; NO: nitric oxide; P: phosphorous; RME: resting metabolic expenditure; Zn: zinc; ↑: increases; were identified under glutamine: nitrogen balance; wound healing time; length of hospital stay and patient mortality; lactulose/mannitol ratio; C-reactive protein; cytokines (IL-6 levels, TNFα levels) and T-cell lymphocytes.

Collagen Deposition (Hydroxyproline Content)0892
Several studies have demonstrated that supplementation with arginine increases collagen deposition and, therefore, enhances wound-breaking strength. The wound-breaking strength is the force needed to disrupt a wound [67]. Barbul et al. [59] observed this improvement in a randomized, controlled trial (RCT) in 36 healthy and non-smoking humans by supplementing their diet with 24.8 g of free arginine as arginine hydrochloride and 17 g of free arginine as arginine aspartate per day for 2 weeks. Hydroxyproline content was assessed as an index of the synthesis and deposition of new collagen in a polytetrafluoroethylene tube inserted in the wound site. An enhanced collagen deposition at 137 and 74% was noted in the arginine hydrochloride (p = 0.028; 23.85 ± 2.16 nmol/cm) and arginine aspartate (p = 0.028, 17.57 ± 2.16 nmol/cm) groups, respectively, following a significant difference observed in the controlled group (p < 0.001; 10.1 ± 2.32 nmol/cm in controls). These results were confirmed later by Nussbaum [64], who carried out a similar trial in 45 healthy elderly people, randomly supplemented or not with 17 g of arginine per day for 14 days. An improvement was also observed in collagen synthesis through the hydroxyproline deposition (17.4 ± 2 nmol/cm; p < 0.02) and T-cell-mediated immune function.
Arginine also influences the nitrogen balance. Nevertheless, this balance improvement has been reported in many, but not in all studies. In a randomized double-blind controlled study by Debats et al. [65], specific parameters related to wound healing were measured after supplementing 30 g of arginine (n = 16) or placebo (n = 19) for 10 days. Angiogenesis, assessed as the number of vessels per high power field significantly increased on day 10 (8.9 ± 3.1 in the arginine group vs. 8 ± 2.8 in the placebo group, p < 0.001).
does not increase or decrease.

Collagen Deposition (Hydroxyproline Content)
Several studies have demonstrated that supplementation with arginine increases collagen deposition and, therefore, enhances wound-breaking strength. The wound-breaking strength is the force needed to disrupt a wound [67]. Barbul et al. [59] observed this improvement in a randomized, controlled trial (RCT) in 36 healthy and non-smoking humans by supplementing their diet with 24.8 g of free arginine as arginine hydrochloride and 17 g of free arginine as arginine aspartate per day for 2 weeks. Hydroxyproline content was assessed as an index of the synthesis and deposition of new collagen in a polytetrafluoroethylene tube inserted in the wound site. An enhanced collagen deposition at 137 and 74% was noted in the arginine hydrochloride (p = 0.028; 23.85 ± 2.16 nmol/cm) and arginine aspartate (p = 0.028, 17.57 ± 2.16 nmol/cm) groups, respectively, following a significant difference observed in the controlled group (p < 0.001; 10.1 ± 2.32 nmol/cm in controls). These results were confirmed later by Nussbaum [64], who carried out a similar trial in 45 healthy elderly people, randomly supplemented or not with 17 g of arginine per day for 14 days. An improvement was also observed in collagen synthesis through the hydroxyproline deposition (17.4 ± 2 nmol/cm; p < 0.02) and T-cell-mediated immune function.
Arginine also influences the nitrogen balance. Nevertheless, this balance improvement has been reported in many, but not in all studies. In a randomized double-blind controlled study by Debats et al. [65], specific parameters related to wound healing were measured after supplementing 30 g of arginine (n = 16) or placebo (n = 19) for 10 days. Angiogenesis, assessed as the number of vessels per high power field significantly increased on day 10 (8.9 ± 3.1 in the arginine group vs. 8 ± 2.8 in the placebo group, p < 0.001). Meanwhile, the increment in re-epithelialization failed to reach significance (85 ± 7.1% in the arginine group vs. 81 ± 8.5% in the placebo group, p > 0.05).
In a double-blind RCT conducted and involving 30 adults for 7 days by Sigal et al. [66], the enhancement of lymphocyte proliferation was not observed (p > 0.05) after intravenous supplementation with 14.7 g of arginine compared to controls, treated with Travasol 10% (an isonitrogenous mix of amino acids). The nitrogen balance measured in the supplemented group (−8.8 g/day) was comparable to the control group (−9.2 g/day, p > 0.05).
Langkamp-Henken et al. [60], also conducted an RCT on 33 elderly patients supplemented with different amounts of arginine (0, 8.5, or 17 g) for 4 weeks. These amounts of arginine used represented approximately 2.2 and 4.5% of an 1800 kcal intake, respectively. The mean daily intake of the patients in this study ranged between 1713 and 2474 kcal with an additional 2.4 and 3.3 g of arginine. After the treatment, no increase in lymphocyte proliferation was observed between groups, while NO increased, although not statistically significant (p > 0.05).
Effects of arginine supplementation on collagen deposition, measured by hydroxyproline content were reported in 2 studies. The study conducted by Barbul et al. [59] reported two results due to two different amounts of supplementation given (17 and 24.8 g arginine). Compared to control, arginine supplementation significantly enhanced hydroxyproline content (MD: 4.49, 95% CI: 3.54, 5.45, p < 0.00001, Figure 8). There was a high heterogeneity (I 2 = 99%) among studies. However, there was a lack of significance between the intervention and control group in the Nussbaum study [64] (MD: −0.01, 95% CI: −1.27, 1.25).
In a double-blind RCT conducted and involving 30 adults for 7 days by Sigal et al. [66], the enhancement of lymphocyte proliferation was not observed (p > 0.05) after intravenous supplementation with 14.7 g of arginine compared to controls, treated with Travasol 10% (an isonitrogenous mix of amino acids). The nitrogen balance measured in the supplemented group (−8.8 g/day) was comparable to the control group (−9.2 g/day, p > 0.05).
Langkamp-Henken et al. [60], also conducted an RCT on 33 elderly patients supplemented with different amounts of arginine (0, 8.5, or 17 g) for 4 weeks. These amounts of arginine used represented approximately 2.2 and 4.5% of an 1800 kcal intake, respectively. The mean daily intake of the patients in this study ranged between 1713 and 2474 kcal with an additional 2.4 and 3.3 g of arginine. After the treatment, no increase in lymphocyte proliferation was observed between groups, while NO increased, although not statistically significant (p > 0.05).
Effects of arginine supplementation on collagen deposition, measured by hydroxyproline content were reported in 2 studies. The study conducted by Barbul et al. [59] reported two results due to two different amounts of supplementation given (17 and 24.8 g arginine). Compared to control, arginine supplementation significantly enhanced hydroxyproline content (MD: 4.49, 95% CI: 3.54, 5.45, p < 0.00001, Figure 8). There was a high heterogeneity (I 2 = 99%) among studies. However, there was a lack of significance between the intervention and control group in the Nussbaum study [64] (MD: −0.01, 95% CI: −1.27, 1.25).

Glutamine (Gln)
Glutamine has been shown to influence several parameters involved in wound healing. This behavior was confirmed by many of the randomized controlled trials that will be reported below, where patients with a total, partial parenteral or early enteral nutrition were supplemented with alanine-glutamine dipeptide, in a concentration ranging from 0.2 to 0.5 g Ala-Gln/kg/day for up to 14 days, as summarized in Table 3. Ala-Gln dipeptide is usually used instead of free Gln, due to its heat stability and its rapid hydrolyzation to free amino acids in plasma [68].

Glutamine (Gln)
Glutamine has been shown to influence several parameters involved in wound healing. This behavior was confirmed by many of the randomized controlled trials that will be reported below, where patients with a total, partial parenteral or early enteral nutrition were supplemented with alanine-glutamine dipeptide, in a concentration ranging from 0.2 to 0.5 g Ala-Gln/kg/day for up to 14 days, as summarized in Table 3. Ala-Gln dipeptide is usually used instead of free Gln, due to its heat stability and its rapid hydrolyzation to free amino acids in plasma [68]. Several studies have demonstrated that supplementation with argin lagen deposition and, therefore, enhances wound-breaking strength. T ing strength is the force needed to disrupt a wound [67]. Barbul et al. [ improvement in a randomized, controlled trial (RCT) in 36 healthy and mans by supplementing their diet with 24.8 g of free arginine as argini and 17 g of free arginine as arginine aspartate per day for 2 weeks. Hyd tent was assessed as an index of the synthesis and deposition of new c tetrafluoroethylene tube inserted in the wound site. An enhanced colla 137 and 74% was noted in the arginine hydrochloride (p = 0.028; 23.85 ± 2 arginine aspartate (p = 0.028, 17.57 ± 2.16 nmol/cm) groups, respectively nificant difference observed in the controlled group (p < 0.001; 10.1 ± 2.32 trols). These results were confirmed later by Nussbaum [64], who car trial in 45 healthy elderly people, randomly supplemented or not with 17 day for 14 days. An improvement was also observed in collagen synth hydroxyproline deposition (17.4 ± 2 nmol/cm; p < 0.02) and T-cell-media tion.
Arginine also influences the nitrogen balance. Nevertheless, this ment has been reported in many, but not in all studies. In a randomi controlled study by Debats et al. [65], specific parameters related to wo measured after supplementing 30 g of arginine (n = 16) or placebo (n Angiogenesis, assessed as the number of vessels per high power field creased on day 10 (8.9 ± 3.1 in the arginine group vs. 8  Several studies have demonstrated that supplementation with arginine increases c lagen deposition and, therefore, enhances wound-breaking strength. The wound-bre ing strength is the force needed to disrupt a wound [67]. Barbul et al. [59] observed t improvement in a randomized, controlled trial (RCT) in 36 healthy and non-smoking mans by supplementing their diet with 24.8 g of free arginine as arginine hydrochlor and 17 g of free arginine as arginine aspartate per day for 2 weeks. Hydroxyproline c tent was assessed as an index of the synthesis and deposition of new collagen in a po tetrafluoroethylene tube inserted in the wound site. An enhanced collagen deposition 137 and 74% was noted in the arginine hydrochloride (p = 0.028; 23.85 ± 2.16 nmol/cm) a arginine aspartate (p = 0.028, 17.57 ± 2.16 nmol/cm) groups, respectively, following a s nificant difference observed in the controlled group (p < 0.001; 10.1 ± 2.32 nmol/cm in c trols). These results were confirmed later by Nussbaum [64], who carried out a sim trial in 45 healthy elderly people, randomly supplemented or not with 17 g of arginine day for 14 days. An improvement was also observed in collagen synthesis through hydroxyproline deposition (17.4 ± 2 nmol/cm; p < 0.02) and T-cell-mediated immune fu tion.
Arginine also influences the nitrogen balance. Nevertheless, this balance impro ment has been reported in many, but not in all studies. In a randomized double-bl controlled study by Debats et al. [65], specific parameters related to wound healing w measured after supplementing 30 g of arginine (n = 16) or placebo (n = 19) for 10 da Angiogenesis, assessed as the number of vessels per high power field significantly creased on day 10 (8.9 ± 3.1 in the arginine group vs. 8 ± 2.8 in the placebo group, p < 0.00 Several studies have demonstrated that supplementation with arginine inc lagen deposition and, therefore, enhances wound-breaking strength. The wou ing strength is the force needed to disrupt a wound [67]. Barbul et al. [59] ob improvement in a randomized, controlled trial (RCT) in 36 healthy and non-sm mans by supplementing their diet with 24.8 g of free arginine as arginine hyd and 17 g of free arginine as arginine aspartate per day for 2 weeks. Hydroxyp tent was assessed as an index of the synthesis and deposition of new collagen tetrafluoroethylene tube inserted in the wound site. An enhanced collagen de 137 and 74% was noted in the arginine hydrochloride (p = 0.028; 23.85 ± 2.16 nm arginine aspartate (p = 0.028, 17.57 ± 2.16 nmol/cm) groups, respectively, follow nificant difference observed in the controlled group (p < 0.001; 10.1 ± 2.32 nmol/ trols). These results were confirmed later by Nussbaum [64], who carried ou trial in 45 healthy elderly people, randomly supplemented or not with 17 g of a day for 14 days. An improvement was also observed in collagen synthesis th hydroxyproline deposition (17.4 ± 2 nmol/cm; p < 0.02) and T-cell-mediated imm tion.
Arginine also influences the nitrogen balance. Nevertheless, this balance ment has been reported in many, but not in all studies. In a randomized do controlled study by Debats et al. [65], specific parameters related to wound he measured after supplementing 30 g of arginine (n = 16) or placebo (n = 19) fo Angiogenesis, assessed as the number of vessels per high power field signif creased on day 10 (8.9 ± 3.1 in the arginine group vs. 8  Several studies have demonstrated that supplementation with argi lagen deposition and, therefore, enhances wound-breaking strength. T ing strength is the force needed to disrupt a wound [67]. Barbul et al. improvement in a randomized, controlled trial (RCT) in 36 healthy and mans by supplementing their diet with 24.8 g of free arginine as argin and 17 g of free arginine as arginine aspartate per day for 2 weeks. Hy tent was assessed as an index of the synthesis and deposition of new c tetrafluoroethylene tube inserted in the wound site. An enhanced colla 137 and 74% was noted in the arginine hydrochloride (p = 0.028; 23.85 ± arginine aspartate (p = 0.028, 17.57 ± 2.16 nmol/cm) groups, respectivel nificant difference observed in the controlled group (p < 0.001; 10.1 ± 2.3 trols). These results were confirmed later by Nussbaum [64], who car trial in 45 healthy elderly people, randomly supplemented or not with 1 day for 14 days. An improvement was also observed in collagen synt hydroxyproline deposition (17.4 ± 2 nmol/cm; p < 0.02) and T-cell-media tion.
Arginine also influences the nitrogen balance. Nevertheless, this ment has been reported in many, but not in all studies. In a random controlled study by Debats et al. [65], specific parameters related to wo measured after supplementing 30 g of arginine (n = 16) or placebo (n Angiogenesis, assessed as the number of vessels per high power fiel creased on day 10 (8.9 ± 3.1 in the arginine group vs. 8  Several studies have demonstrated that supplementation with arginine increases collagen deposition and, therefore, enhances wound-breaking strength. The wound-breaking strength is the force needed to disrupt a wound [67]. Barbul et al. [59] observed this improvement in a randomized, controlled trial (RCT) in 36 healthy and non-smoking humans by supplementing their diet with 24.8 g of free arginine as arginine hydrochloride and 17 g of free arginine as arginine aspartate per day for 2 weeks. Hydroxyproline content was assessed as an index of the synthesis and deposition of new collagen in a polytetrafluoroethylene tube inserted in the wound site. An enhanced collagen deposition at 137 and 74% was noted in the arginine hydrochloride (p = 0.028; 23.85 ± 2.16 nmol/cm) and arginine aspartate (p = 0.028, 17.57 ± 2.16 nmol/cm) groups, respectively, following a significant difference observed in the controlled group (p < 0.001; 10.1 ± 2.32 nmol/cm in controls). These results were confirmed later by Nussbaum [64], who carried out a similar trial in 45 healthy elderly people, randomly supplemented or not with 17 g of arginine per day for 14 days. An improvement was also observed in collagen synthesis through the hydroxyproline deposition (17.4 ± 2 nmol/cm; p < 0.02) and T-cell-mediated immune function.
Arginine also influences the nitrogen balance. Nevertheless, this balance improvement has been reported in many, but not in all studies. In a randomized double-blind controlled study by Debats et al. [65], specific parameters related to wound healing were measured after supplementing 30 g of arginine (n = 16) or placebo (n = 19) for 10 days. Angiogenesis, assessed as the number of vessels per high power field significantly increased on day 10 (8.9 ± 3.1 in the arginine group vs. 8 ± 2.8 in the placebo group, p < 0.001). Research on the pharmacological effects of arginine supplementat mostly based on its use for acute wounds, although some trials have studie chronic wounds (Table 2). Several studies have demonstrated that supplementation with arginine lagen deposition and, therefore, enhances wound-breaking strength. The ing strength is the force needed to disrupt a wound [67]. Barbul et al. [59] improvement in a randomized, controlled trial (RCT) in 36 healthy and non mans by supplementing their diet with 24.8 g of free arginine as arginine h and 17 g of free arginine as arginine aspartate per day for 2 weeks. Hydrox tent was assessed as an index of the synthesis and deposition of new colla tetrafluoroethylene tube inserted in the wound site. An enhanced collagen 137 and 74% was noted in the arginine hydrochloride (p = 0.028; 23.85 ± 2.16 arginine aspartate (p = 0.028, 17.57 ± 2.16 nmol/cm) groups, respectively, fo nificant difference observed in the controlled group (p < 0.001; 10.1 ± 2.32 nm trols). These results were confirmed later by Nussbaum [64], who carried trial in 45 healthy elderly people, randomly supplemented or not with 17 g o day for 14 days. An improvement was also observed in collagen synthesi hydroxyproline deposition (17.4 ± 2 nmol/cm; p < 0.02) and T-cell-mediated tion.
Arginine also influences the nitrogen balance. Nevertheless, this bala ment has been reported in many, but not in all studies. In a randomized controlled study by Debats et al. [65], specific parameters related to wound measured after supplementing 30 g of arginine (n = 16)  were identified under glutamine: nitrogen balance; wound healing time; length of hospital stay and patient mortality; lactulose/mannitol ratio; C-reactive protein; cytokines (IL-6 levels, TNFα levels) and T-cell lymphocytes.

Collagen Deposition (Hydroxyproline Content)0892
Several studies have demonstrated that supplementation with arginine increases collagen deposition and, therefore, enhances wound-breaking strength. The wound-breaking strength is the force needed to disrupt a wound [67]. Barbul et al. [59] observed this improvement in a randomized, controlled trial (RCT) in 36 healthy and non-smoking humans by supplementing their diet with 24.8 g of free arginine as arginine hydrochloride and 17 g of free arginine as arginine aspartate per day for 2 weeks. Hydroxyproline content was assessed as an index of the synthesis and deposition of new collagen in a polytetrafluoroethylene tube inserted in the wound site. An enhanced collagen deposition at 137 and 74% was noted in the arginine hydrochloride (p = 0.028; 23.85 ± 2.16 nmol/cm) and arginine aspartate (p = 0.028, 17.57 ± 2.16 nmol/cm) groups, respectively, following a significant difference observed in the controlled group (p < 0.001; 10.1 ± 2.32 nmol/cm in controls). These results were confirmed later by Nussbaum [

Collagen Deposition (Hydroxyproline Content)0892
Several studies have demonstrated that supplementation with arginine increases collagen deposition and, therefore, enhances wound-breaking strength. The wound-breaking strength is the force needed to disrupt a wound [67]. Barbul et al. [59] observed this improvement in a randomized, controlled trial (RCT) in 36 healthy and non-smoking humans by supplementing their diet with 24.8 g of free arginine as arginine hydrochloride and 17 g of free arginine as arginine aspartate per day for 2 weeks. Hydroxyproline content was assessed as an index of the synthesis and deposition of new collagen in a polytetrafluoroethylene tube inserted in the wound site. An enhanced collagen deposition at 137 and 74% was noted in the arginine hydrochloride (p = 0.028; 23.85 ± 2.16 nmol/cm) and arginine aspartate (p = 0.028, 17.57 ± 2.16 nmol/cm) groups, respectively, following a significant difference observed in the controlled group (p < 0.001; 10.1 ± 2.32 nmol/cm in controls). These results were confirmed later by Nussbaum [64], who carried out a similar trial in 45 healthy elderly people, randomly supplemented or not with 17 g of arginine per day for 14 days. An improvement was also observed in collagen synthesis through the hydroxyproline deposition (17.4 ± 2 nmol/cm; p < 0.02) and T-cell-mediated immune function.
Arginine also influences the nitrogen balance. Nevertheless, this balance improvement has been reported in many, but not in all studies. In a randomized double-blind controlled study by Debats et al. [65], specific parameters related to wound healing were LOS, ↑ NO, ↓ Intestinal permeability Sahin et al. [88] 10.5 ± 3.6 days Critical illness 40 0.3 0.45 Isonitrogenous solution levels, TNFα levels) and T-cell lymphocytes.

Collagen Deposition (Hydroxyproline Content)0892
Several studies have demonstrated that supplementation with arginine increases collagen deposition and, therefore, enhances wound-breaking strength. The wound-breaking strength is the force needed to disrupt a wound [67]. Barbul et al. [59] observed this improvement in a randomized, controlled trial (RCT) in 36 healthy and non-smoking humans by supplementing their diet with 24.8 g of free arginine as arginine hydrochloride and 17 g of free arginine as arginine aspartate per day for 2 weeks. Hydroxyproline content was assessed as an index of the synthesis and deposition of new collagen in a polytetrafluoroethylene tube inserted in the wound site. An enhanced collagen deposition at 137 and 74% was noted in the arginine hydrochloride (p = 0.028; 23.85 ± 2.16 nmol/cm) and arginine aspartate (p = 0.028, 17.57 ± 2.16 nmol/cm) groups, respectively, following a significant difference observed in the controlled group (p < 0.001; 10.1 ± 2.32 nmol/cm in controls). These results were confirmed later by Nussbaum [64], who carried out a similar trial in 45 healthy elderly people, randomly supplemented or not with 17 g of arginine per day for 14 days. An improvement was also observed in collagen synthesis through the hydroxyproline deposition (17.4 ± 2 nmol/cm; p < 0.02) and T-cell-mediated immune function.
Arginine also influences the nitrogen balance. Nevertheless, this balance improve- were identified under glutamine: nitrogen balance; wound healing time; length of hospi tal stay and patient mortality; lactulose/mannitol ratio; C-reactive protein; cytokines (ILlevels, TNFα levels) and T-cell lymphocytes.

Arginine (Arg)
Research on the pharmacological effects of arginine supplementation has been mostly based on its use for acute wounds, although some trials have studied its effect on chronic wounds (Table 2). Several studies have demonstrated that supplementation with arginine increases col lagen deposition and, therefore, enhances wound-breaking strength. The wound-break ing strength is the force needed to disrupt a wound [67]. Barbul et al. [59] observed thi improvement in a randomized, controlled trial (RCT) in 36 healthy and non-smoking hu mans by supplementing their diet with 24.8 g of free arginine as arginine hydrochloride and 17 g of free arginine as arginine aspartate per day for 2 weeks. Hydroxyproline con tent was assessed as an index of the synthesis and deposition of new collagen in a poly tetrafluoroethylene tube inserted in the wound site. An enhanced collagen deposition a 137 and 74% was noted in the arginine hydrochloride (p = 0.028; 23.85 ± 2.16 nmol/cm) and arginine aspartate (p = 0.028, 17.57 ± 2.16 nmol/cm) groups, respectively, following a sig nificant difference observed in the controlled group (p < 0.001; 10.1 ± 2.32 nmol/cm in con trols). These results were confirmed later by Nussbaum [64], who carried out a simila were identified under glutamine: nitrogen balance; wound healing time; len tal stay and patient mortality; lactulose/mannitol ratio; C-reactive protein; cy levels, TNFα levels) and T-cell lymphocytes.

Arginine (Arg)
Research on the pharmacological effects of arginine supplementati mostly based on its use for acute wounds, although some trials have studied chronic wounds (Table 2). Several studies have demonstrated that supplementation with arginine lagen deposition and, therefore, enhances wound-breaking strength. The w ing strength is the force needed to disrupt a wound [67]. Barbul   Several studies have demonstrated that supplementation with arginine increases co lagen deposition and, therefore, enhances wound-breaking strength. The wound-break ing strength is the force needed to disrupt a wound [67]. Barbul et al. [59] observed thi improvement in a randomized, controlled trial (RCT) in 36 healthy and non-smoking hu mans by supplementing their diet with 24.8 g of free arginine as arginine hydrochlorid and 17 g of free arginine as arginine aspartate per day for 2 weeks. Hydroxyproline con tent was assessed as an index of the synthesis and deposition of new collagen in a poly tetrafluoroethylene tube inserted in the wound site. An enhanced collagen deposition a 137 and 74% was noted in the arginine hydrochloride (p = 0.028; 23.85 ± 2.16 nmol/cm) an arginine aspartate (p = 0.028, 17.57 ± 2.16 nmol/cm) groups, respectively, following a sig nificant difference observed in the controlled group (p < 0.001; 10.1 ± 2.32 nmol/cm in con trols). These results were confirmed later by Nussbaum [64], who carried out a simila trial in 45 healthy elderly people, randomly supplemented or not with 17 g of arginine pe day for 14 days. An improvement was also observed in collagen synthesis through th hydroxyproline deposition (17.4 ± 2 nmol/cm; p < 0.02) and T-cell-mediated immune func tion.
Arginine also influences the nitrogen balance. Nevertheless, this balance improve ment has been reported in many, but not in all studies. In a randomized double-blin controlled study by Debats et al. [65], specific parameters related to wound healing wer measured after supplementing 30 g of arginine (n = 16) or placebo (n = 19) for 10 days Angiogenesis, assessed as the number of vessels per high power field significantly in creased on day 10 (8.9 ± 3.1 in the arginine group vs. 8  Several studies have demonstrated that supplementation with arginine increases co lagen deposition and, therefore, enhances wound-breaking strength. The wound-brea ing strength is the force needed to disrupt a wound [67]. Barbul et al. [59] observed th improvement in a randomized, controlled trial (RCT) in 36 healthy and non-smoking h mans by supplementing their diet with 24.8 g of free arginine as arginine hydrochlorid and 17 g of free arginine as arginine aspartate per day for 2 weeks. Hydroxyproline co tent was assessed as an index of the synthesis and deposition of new collagen in a pol tetrafluoroethylene tube inserted in the wound site. An enhanced collagen deposition 137 and 74% was noted in the arginine hydrochloride (p = 0.028; 23.85 ± 2.16 nmol/cm) an arginine aspartate (p = 0.028, 17.57 ± 2.16 nmol/cm) groups, respectively, following a si nificant difference observed in the controlled group (p < 0.001; 10.1 ± 2.32 nmol/cm in co trols). These results were confirmed later by Nussbaum [64], who carried out a simil trial in 45 healthy elderly people, randomly supplemented or not with 17 g of arginine p day for 14 days. An improvement was also observed in collagen synthesis through th hydroxyproline deposition (17.4 ± 2 nmol/cm; p < 0.02) and T-cell-mediated immune fun tion.
Arginine also influences the nitrogen balance. Nevertheless, this balance improv ment has been reported in many, but not in all studies. In a randomized double-blin controlled study by Debats et al. [65], specific parameters related to wound healing we measured after supplementing 30 g of arginine (n = 16) or placebo (n = 19) for 10 day Angiogenesis, assessed as the number of vessels per high power field significantly i creased on day 10 (8.9 ± 3.1 in the arginine group vs. 8  were identified under glutamine: nitrogen balance; wound healing time; length of hospital stay and patient mortality; lactulose/mannitol ratio; C-reactive protein; cytokines (IL-6 levels, TNFα levels) and T-cell lymphocytes.

Collagen Deposition (Hydroxyproline Content)0892
Several studies have demonstrated that supplementation with arginine increases collagen deposition and, therefore, enhances wound-breaking strength. The wound-breaking strength is the force needed to disrupt a wound [67]. Barbul et al. [59] observed this improvement in a randomized, controlled trial (RCT) in 36 healthy and non-smoking humans by supplementing their diet with 24.8 g of free arginine as arginine hydrochloride and 17 g of free arginine as arginine aspartate per day for 2 weeks. Hydroxyproline content was assessed as an index of the synthesis and deposition of new collagen in a polytetrafluoroethylene tube inserted in the wound site. An enhanced collagen deposition at 137 and 74% was noted in the arginine hydrochloride (p = 0.028; 23.85 ± 2.16 nmol/cm) and arginine aspartate (p = 0.028, 17.57 ± 2.16 nmol/cm) groups, respectively, following a significant difference observed in the controlled group (p < 0.001; 10.1 ± 2.32 nmol/cm in controls). These results were confirmed later by Nussbaum [64], who carried out a similar trial in 45 healthy elderly people, randomly supplemented or not with 17 g of arginine per day for 14 days. An improvement was also observed in collagen synthesis through the does not increase or decrease.

Nitrogen Balance
In two double-blind RCT performed in 48 patients supplemented with 0.28 g/kg/day Gln for 6 days, by Lin et al. [79,85], higher nitrogen balance, although not significant (−3.2 ± 1.6 vs. −6.5 ± 2.7 g N, p > 0.05) was observed after Gln treatment. Cumulative nitrogen balance was higher in Glu-supplemented patients with lower illness severity, assessed with APACHE II scores (acute physiology score + age points + chronic health points), but this was not observed when severity was higher (APACHE II score > 6). However, when compared with controls, the reducing effect was not reported, showing that the improving effect on cumulative nitrogen balance is not due to the reduction of muscle protein breakdown, but an enhanced protein synthesis.
In contrast, a higher cumulative nitrogen balance, adjusted to standard body surface area, was not noted (−193 ± 50 vs. −198 ± 77 g N, p > 0.05) in a prospective doubleblind RCT conducted by Duška et al. [90], who supplemented 30 patients suffering from critical illnesses with 0.2 g/kg/day Gln for 13 days, and compared the results against the placebo group, treated with isonitrogenous nutrition. Similar results were reported by Mertes et al. [75] in a double-blind RCT on 37 patients undergoing major abdominal surgery for 5 days (−14.1 ± 9.1 vs. −31.7 ± 11.4 g N, p < 0.05). Jiang et al. [73] also noted a rise of the cumulative nitrogen balance (144.3 ± 145.6 vs. −5.1 ± 162.7 mg/kg, p = 0.0004) in a double-blind RCT in 60 patients undergoing abdominal surgery, supplemented with 0.34 g/kg/day or an isonitrogenous solution for 7 days.

Nitrogen Balance
In two double-blind RCT performed in 48 patients supplemented with 0.28 g/kg/day Gln for 6 days, by Lin et al. [79,85], higher nitrogen balance, although not significant (−3.2 ± 1.6 vs. −6.5 ± 2.7 g N, p > 0.05) was observed after Gln treatment. Cumulative nitrogen balance was higher in Glu-supplemented patients with lower illness severity, assessed with APACHE II scores (acute physiology score + age points + chronic health points), but this was not observed when severity was higher (APACHE II score > 6). However, when compared with controls, the reducing effect was not reported, showing that the improving effect on cumulative nitrogen balance is not due to the reduction of muscle protein breakdown, but an enhanced protein synthesis.
In contrast, a higher cumulative nitrogen balance, adjusted to standard body surface area, was not noted (−193 ± 50 vs. −198 ± 77 g N, p > 0.05) in a prospective double-blind RCT conducted by Duška et al. [90], who supplemented 30 patients suffering from critical illnesses with 0.2 g/kg/day Gln for 13 days, and compared the results against the placebo group, treated with isonitrogenous nutrition. Similar results were reported by Mertes et al. [75] in a double-blind RCT on 37 patients undergoing major abdominal surgery for 5 days (−14.1 ± 9.1 vs. −31.7 ± 11.4 g N, p < 0.05). Jiang et al. [73] also noted a rise of the cumulative nitrogen balance (144.3 ± 145.6 vs. −5.1 ± 162.7 mg/kg, p = 0.0004) in a doubleblind RCT in 60 patients undergoing abdominal surgery, supplemented with 0.34 g/kg/day or an isonitrogenous solution for 7 days.

Wound Healing Time
Two trials [72,82] measured the effect of glutamine supplementation on wound healing. Zhou et al. [82] performed a double-blind RCT and noticed a lower infection rate (13 vs. 26%), although this difference failed to reach significance (p > 0.05). Also, it was noted a significant reduction of the wound healing time in the study group (32.1 ± 3.3 days) compared to the control group (36.6 ± 6.6 days, p < 0.012) after supplementing 30 patients suffering from severe burns with 0.34 g/kg/day Gln or an isonitrogenous solution, for 12

Wound Healing Time
Two trials [72,82] measured the effect of glutamine supplementation on wound healing. Zhou et al. [82] performed a double-blind RCT and noticed a lower infection rate (13 vs. 26%), although this difference failed to reach significance (p > 0.05). Also, it was noted a significant reduction of the wound healing time in the study group (32.1 ± 3.3 days) compared to the control group (36.6 ± 6.6 days, p < 0.012) after supplementing 30 patients suffering from severe burns with 0.34 g/kg/day Gln or an isonitrogenous solution, for 12 days. This data is in agreement with the RCT performed by Jacobi et al. [72] where, after supplementing 34 patients with 0.27 g/kg/day Gln for 7 days, observed a signifi-cant increase in wound healing (p < 0.05), explained in part by the incidence of wound complications post-surgery.

Length of Hospital Stay (LOS) and Patient mortality
The decrease in LOS was reported by many studies. Neri et al. [77] studied the effect of 0.2 g/kg/day Gln in 33 patients supplemented for at least 7 days. In this trial, the study group spent 11.5 ± 2 days hospitalized, versus the control group, which spent 15 ± 3 days (p < 0.05). Meanwhile, in the prospective double-blind RTC carried out by Yao et al. [86] in 40 people for 5 days, the study group spent 10.6 ± 1.2 days in the hospital, while the non-supplemented group spent 11.7 ± 2 days (p = 0.03).
These data are in agreement with Fan et al. [96], who performed an RCT in 40 patients undergoing surgery and observed a trend to a reduction both in LOS (22.3 ± 2.1 vs. 24.9 ± 1.7 days, p = 0.32) and infectious complications after supplementation with 0.13 g/kg/day Gln, although these data failed to reach significance. The reduction in LOS and mortality was also observed by Wischmeyer et al. [78] in a double-blind RCT in 31 patients with severe burns, supplemented with 0.57 g/kg/day L-Gln for at least 7 days via parenteral feeding. However, in this case, there was no significant difference seen in the LOS (31 ± 10.1 vs. 30 ± 9.3, p > 0.05) nor mortality (1 vs. 4 deaths, p = 0.19).
Goeters et al. [61] also noticed a patient mortality decline within 6 months (11/33 vs. 21/35 deaths, p < 0.05), resulting in a 66.7% increase of survival of patients treated for ≥ 9 days, compared to 40% in controls, in an unblinded parallel multicentre RCT in 144 critically ill patients supplemented with 0.2 g/kg/day Gln.
Karwowska et al. [76] studied the effect of glutamine supplementation and observed a decrease in LOS; from 15.1 ± 3 days to 12.5 ± 1.2 days (p = 0.005) in the supplemented group. The reduction in LOS was also seen in a double-blind RCT carried out by Powell-Tuck et al. [74] in 168 patients, supplemented with 20 g Gln as part of the nitrogen source of parenteral nutrition or standard feeds, for 7 days. The study group spent an average of 15 fewer days in hospital, compared with the control group (46 ± 10.7 vs. 30 ± 7.2 days, p < 0.03). The latter study failed to demonstrate a decrease in morbidity and mortality in the study group, probably due to the younger age of the patients (48 ± 17 years old vs. 80 ± 19 years old). Nevertheless, this limitation was avoided in a study by Xian-Li et al. [62] in a non-blinded RCT in 69 patients suffering from critical illnesses for 14 days, and it was seen, not only a reduction in LOS (25.3 ± 7.6 vs. 39.1 ± 10.6 days, p < 0.01), but also in patient mortality (0 vs. 43.5%, p < 0.01) after supplementing 0.2 g/kg/day Gln. The same results were obtained by Wernerman et al. [102] in a double-blind RCT, observing a reduction of patient mortality in ICU (8/11 vs. 14/20, p < 0.05) after supplementing 413 patients, in ICU, with 0.28 g/kg/day Gln for 7 days.
On the other hand, Ockenga et al. [80] reported a significant reduction in LOS in a double-blind RCT in 28 patients suffering from acute pancreatitis supplemented with 0.2 g/kg/day Gln for at least 7 days. In this trial, the LOS was an average of 4 days shorter in the study group (21 days) compared to the control group (25 days, p < 0.05) despite the small sample studied. In the trial conducted by Mertes et al. [75] a decrease in hospital stay was also noted (12.8 ± 2.6 vs. 17.5 ± 6.4 days, p < 0.05) compared to the control group. Similar results were observed by Jiang et al. [73] who noted a decrease in 4 days of LOS (12.5 ± 5.1 vs. 16.4 ± 7.1 days, p = 0.02). Morlion et al. [71] not only noted the reduction in LOS (15.5 ± 0.7 vs. 21.7± 2.8 days, p < 0.05), but there were also evident improvements both in mood and general well-being in the supplemented patients.
On the contrary, in a prospective double-blind RCT performed by Déchelotte et al. [87] in 114 patients with critical illnesses treated with 0.33 g/kg/day Gln, no significant differences in LOS nor patient mortality were observed (1.9 vs. 3.8% deaths, p > 0.05), although a reduction of the incidence of infectious complications (39 vs. 64%, p < 0.02) was noted. The decrease of nosocomial infections, such as urinary tract infections (2.3 vs. 16.9%, p = 0.03) and pneumonia (8 vs. 29%, p = 0.02) were also observed by Grau et al. [100]. This trial studied the effect of 0.35 g/kg/day Gln in a multicentre, prospective, double-blind, RCT in 502 patients admitted to the UCI. In another double-blind RCT performed by Estívariz et al. [91] in 63 people undergoing surgery and in ICU, patients were supplemented with 0.34 g/kg/day Gln and a lessening of nosocomial infections was observed (13 vs. 36 cases, p < 0.03). Kłek et al. [84] also reported a decrease in postoperative complications (23.3 vs. 36.6%, p < 0.05) and LOS (14.8 vs. 16.4 days, p < 0.05) in an RCT performed in 69 patients undergoing surgery supplemented with 0.27 g/kg/day Gln for 12 days.
On the contrary, a double-blind RCT conducted by Andrews et al. [98], patients supplemented with 0.2 g/kg/day Gln for at least 5 days, neither showed a significant improvement in the incidence of infections nor patient mortality compared to the control group (p > 0.05).
The effect of glutamine on LOS was reported by 21 studies involving 1042 participants. According to the meta-analysis, glutamine supplementation has shown to significantly decrease the days of hospitalization (MD: −2.65, 95% CI: −3.10, −2.21, p < 0.00001, Figure 10). Exhibiting a high heterogeneity among studies (I 2 = 84%). Nevertheless, not all the studies showed a significant effect on this parameter. This is the case of the following studies:  On the contrary, in a prospective double-blind RCT performed by Déchelotte et al. [87] in 114 patients with critical illnesses treated with 0.33 g/kg/day Gln, no significant differences in LOS nor patient mortality were observed (1.9 vs. 3.8% deaths, p > 0.05), although a reduction of the incidence of infectious complications (39 vs. 64%, p < 0.02) was noted. The decrease of nosocomial infections, such as urinary tract infections (2.3 vs. 16.9%, p = 0.03) and pneumonia (8 vs. 29%, p = 0.02) were also observed by Grau et al. [100]. This trial studied the effect of 0.35 g/kg/day Gln in a multicentre, prospective, double-blind, RCT in 502 patients admitted to the UCI. In another double-blind RCT performed by Estívariz et al. [91] in 63 people undergoing surgery and in ICU, patients were supplemented with 0.34 g/kg/day Gln and a lessening of nosocomial infections was observed (13 vs. 36 cases, p < 0.03). Kłek et al. [84] also reported a decrease in postoperative complications (23.3 vs. 36.6%, p < 0.05) and LOS (14.8 vs. 16.4 days, p < 0.05) in an RCT performed in 69 patients undergoing surgery supplemented with 0.27 g/kg/day Gln for 12 days.
On the contrary, a double-blind RCT conducted by Andrews et al. [98], patients supplemented with 0.2 g/kg/day Gln for at least 5 days, neither showed a significant improvement in the incidence of infections nor patient mortality compared to the control group (p > 0.05).
The effect of glutamine on LOS was reported by 21 studies involving 1042 participants. According to the meta-analysis, glutamine supplementation has shown to significantly decrease the days of hospitalization (MD: −2.65, 95% CI: −3.10, −2.21, p < 0.00001, Figure 10). Exhibiting a high heterogeneity among studies (I 2 = 84%). Nevertheless, not all the studies showed a significant effect on this parameter. This is the case of the following studies: Wischmeyer et al. [78]    Overall data from 11 studies involving 696 participants (340 and 356 in the study and control group respectively) were evaluated. Patient mortality occurred less frequently in the glutamine-supplemented group than in controls (46 (13.53%) vs. (89 (25%) participants). The odds ratio was 0.48 (95% CI: 0.32, 0.72, p = 0.0004, Figure 11), and heterogeneity among studies was 14%. The number needed to treat was 10. In other words, 10 patients would need to receive glutamine supplementation to prevent an additional fatality. Overall data from 11 studies involving 696 participants (340 and 356 in the study and control group respectively) were evaluated. Patient mortality occurred less frequently in the glutamine-supplemented group than in controls (46 (13.53%) vs. (89 (25%) participants). The odds ratio was 0.48 (95% CI: 0.32, 0.72, p = 0.0004, Figure 11), and heterogeneity among studies was 14%. The number needed to treat was 10. In other words, 10 patients would need to receive glutamine supplementation to prevent an additional fatality. Five studies involving 210 participants reported data on intestinal permeability, assessed by lactulose/mannitol ratio. L/M significantly decreased in those participants supplemented with glutamine (MD: −0.01, 95% CI: −0.02, −0.01, p < 0.00001, Figure 12). The heterogeneity given by I 2 was 97%.

Lactulose/Mannitol Ratio
Zhou et al. [82] observed a decrease in the intestinal permeability in the study group (0.021 ± 0.006 vs. 0.025 ± 0.007 L/M ratio, p = 0.115). These data are in agreement with those by Jiang et al. [73] who also noted a decrease in intestinal permeability, assessed by lactulose/mannitol excretion rate ratio (L/M ratio) (0.097 ± 0.063 vs. 0.132 ± 0.081 L/M ratio, p = 0.02). Similar results were reported by Xu et al. [103] who also noted the reduction of L/M (p < 0.05).
Five studies involving 210 participants reported data on intestinal permeability, assessed by lactulose/mannitol ratio. L/M significantly decreased in those participants supplemented with glutamine (MD: −0.01, 95% CI: −0.02, −0.01, p < 0.00001, Figure 12). The heterogeneity given by I 2 was 97%. Overall data from 11 studies involving 696 participants (340 and 356 in the study and control group respectively) were evaluated. Patient mortality occurred less frequently in the glutamine-supplemented group than in controls (46 (13.53%) vs. (89 (25%) participants). The odds ratio was 0.48 (95% CI: 0.32, 0.72, p = 0.0004, Figure 11), and heterogeneity among studies was 14%. The number needed to treat was 10. In other words, 10 patients would need to receive glutamine supplementation to prevent an additional fatality. Figure 11. Patient mortality: fixed-effects meta-analysis and forest plot from studies providing supplementation of glutamine.

Lactulose/Mannitol Ratio
Zhou et al. [82] observed a decrease in the intestinal permeability in the study group (0.021 ± 0.006 vs. 0.025 ± 0.007 L/M ratio, p = 0.115). These data are in agreement with those by Jiang et al. [73] who also noted a decrease in intestinal permeability, assessed by lactulose/mannitol excretion rate ratio (L/M ratio) (0.097 ± 0.063 vs. 0.132 ± 0.081 L/M ratio, p = 0.02). Similar results were reported by Xu et al. [103] who also noted the reduction of L/M (p < 0.05).

C-Reactive Protein
Çekmen et al. [99] observed a trend to the reduction in CRP (57.65 ± 41.81 vs. 82.87 ± 69.41 mg/L, p = 0.32) compared to the control group. Nevertheless, these results did not reach significance.
A double-blind RCT performed by Lu et al. [101] on 50 patients, suffering from gastrointestinal cancer and undergoing surgery, supplemented with 0.3 g/kg/day Gln; reported a significant reduction in CRP serum level (16.7 ± 11.8 vs. 35.2 ± 24.8 mg/dL, p = 0.013). Besides, in the control group, there were noted 4 cases of infectious complications while none were observed in the study group (p = 0.037). The reduction in CRP (p < 0.01) was also observed by Wischmeyer et al. [78] in a double-blind RCT in 31 patients with severe burns, supplemented with 0.57 g/kg/day L-Gln for at least 7 days via parenteral feeding. Ockenga et al. [80], as well as Dong et al. [92] also reported a significant decrease in CRP (30 ± 42 vs. 34 ± 51, p < 0.01), ( Eleven studies reported the effect of glutamine supplementation on CRP levels. Although, individually, 5 studies data did not reach statistical significance [63,80,87,88,99], the overall effect showed the reduction of CRP levels after supplementation (MD: −1.10, 95% CI: −1.26, −0.93, p < 0.00001, Figure 13). The heterogeneity among studies was 82%.
A double-blind RCT performed by Lu et al. [101] on 50 patients, suffering from gastrointestinal cancer and undergoing surgery, supplemented with 0.3 g/kg/day Gln; reported a significant reduction in CRP serum level (16.7 ± 11.8 vs. 35.2 ± 24.8 mg/dL, p = 0.013). Besides, in the control group, there were noted 4 cases of infectious complications while none were observed in the study group (p = 0.037). The reduction in CRP (p < 0.01) was also observed by Wischmeyer et al. [78] in a double-blind RCT in 31 patients with severe burns, supplemented with 0.57 g/kg/day L-Gln for at least 7 days via parenteral feeding. Ockenga et al. [80], as well as Dong et al. [92] also reported a significant decrease in CRP ( Eleven studies reported the effect of glutamine supplementation on CRP levels. Although, individually, 5 studies data did not reach statistical significance [63,80,87,88,99], the overall effect showed the reduction of CRP levels after supplementation (MD: −1.10, 95% CI: −1.26, −0.93, p < 0.00001, Figure 13). The heterogeneity among studies was 82%.
On the other hand, Xu et al. [103] noted a reduction in TNFα levels (p = 0.01) in an RCT in 80 patients supplemented with glutamine administered via early enteral nutrition.
O'Riordain et al. [69] performed a double-blind RCT in 22 patients undergoing surgery and supplemented with 0.18 g/kg/day Gln in the form of glycyl-glutamine, for 5 days. This trial measured the IL-6 and TNFα levels. Nevertheless, the data failed to reach significance (p = 0.27 and p > 0.05, respectively). The lack of significance regarding the production of IL-6 was also observed in a double-blind RCT performed by De Beaux et al. [70] in 14 patients suffering from critical illnesses.
On the other hand, Xu et al. [103] noted a reduction in TNFα levels (p = 0.01) in an RCT in 80 patients supplemented with glutamine administered via early enteral nutrition.
O'Riordain et al. [69] performed a double-blind RCT in 22 patients undergoing surgery and supplemented with 0.18 g/kg/day Gln in the form of glycyl-glutamine, for 5 days. This trial measured the IL-6 and TNFα levels. Nevertheless, the data failed to reach significance (p = 0.27 and p > 0.05, respectively). The lack of significance regarding the production of IL-6 was also observed in a double-blind RCT performed by De Beaux et al. [70] in 14 patients suffering from critical illnesses.

Arginine
Based on the findings of the systematic review, arginine supplementation resulted in greater collagen formation assessed by hydroxyproline level (p < 0.00001). The deposition of collagen could in part be enhanced by T-cell-mediated immune function since they recruit and activate fibroblasts which play a key role in wound repair [105].
The effect of arginine supplementation on T-cell lymphocytes and nitrogen balance was reported in some studies. Nevertheless, its beneficial effect was not always observed [66]. A reason that could explain this outcome may be the lack of additional calories administered either parentally or enterally, which attenuates or even eliminates the pharmaceutical effect of arginine [18,19]. This occurs because around 40% of arginine is catabolized in a single pass in the small intestine, by the type II arginase, and to a much lesser extent, by NO synthase [106]. Indeed, Castillo et al. [107] stated that only 0.34% of the arginine intake absorbed in the splanchnic bed is used to synthesize NO, contributing to 16% of the daily production of NO. Attempts to meet energy requirements were done in most trials, with a minimum of 120 kcal/day in the trial conducted by Debats et al. [65] and a maximum of 2474 kcal/day in the study performed by Langkamp-Henken et al. [60]. Some studies did not show a significant increase or decrease in lymphocyte proliferation and nitrogen balance, respectively [60]. The explanation for this could be the timing of the measurements. Since the elimination of half-life of an oral load of arginine is about 80 min [108].

Glutamine
Regarding glutamine supplementation, the greater cumulative nitrogen balance noted in some of these studies explains the use of glutamine by the body as a substrate for the synthesis of NO. Improved nitrogen retention is associated with a shorter length of hospital stay, and high levels of IL-6 are associated with infections and mortality. The lower production of proinflammatory cytokines might also be explained due to the decrease in intestinal permeability [48]. Therefore, an increase in the nitrogen balance and decline of IL-6 may explain why in many studies, there was a reduction in the length of Figure 15. T-cell lymphocytes: fixed-effects meta-analysis and forest plot from studies providing supplementation of glutamine.

Arginine
Based on the findings of the systematic review, arginine supplementation resulted in greater collagen formation assessed by hydroxyproline level (p < 0.00001). The deposition of collagen could in part be enhanced by T-cell-mediated immune function since they recruit and activate fibroblasts which play a key role in wound repair [105].
The effect of arginine supplementation on T-cell lymphocytes and nitrogen balance was reported in some studies. Nevertheless, its beneficial effect was not always observed [66]. A reason that could explain this outcome may be the lack of additional calories administered either parentally or enterally, which attenuates or even eliminates the pharmaceutical effect of arginine [18,19]. This occurs because around 40% of arginine is catabolized in a single pass in the small intestine, by the type II arginase, and to a much lesser extent, by NO synthase [106]. Indeed, Castillo et al. [107] stated that only 0.34% of the arginine intake absorbed in the splanchnic bed is used to synthesize NO, contributing to 16% of the daily production of NO. Attempts to meet energy requirements were done in most trials, with a minimum of 120 kcal/day in the trial conducted by Debats et al. [65] and a maximum of 2474 kcal/day in the study performed by Langkamp-Henken et al. [60]. Some studies did not show a significant increase or decrease in lymphocyte proliferation and nitrogen balance, respectively [60]. The explanation for this could be the timing of the measurements. Since the elimination of half-life of an oral load of arginine is about 80 min [108].

Glutamine
Regarding glutamine supplementation, the greater cumulative nitrogen balance noted in some of these studies explains the use of glutamine by the body as a substrate for the synthesis of NO. Improved nitrogen retention is associated with a shorter length of hospital stay, and high levels of IL-6 are associated with infections and mortality. The lower production of proinflammatory cytokines might also be explained due to the decrease in intestinal permeability [48]. Therefore, an increase in the nitrogen balance and decline of IL-6 may explain why in many studies, there was a reduction in the length of hospitalization and patient mortality observed [61]. However, the decrease in mortality was not always significant in this review, hence not fully agreeing with the review conducted by Bollhalder et al. [53].
In a prospective double-blind RCT performed by Déchelotte et al. [87] in 114 patients with critical illnesses, no significant differences in LOS nor patient mortality were observed (1.9 vs. 3.8% deaths, p > 0.05), and even if there was an improvement in the nitrogen balance, this variation did not reach significance (−2.44 ± 8.6 vs. −4.4 ± 13.2, p > 0.05). In this case, the benefits of supplementation may have been due to the decrease in intestinal permeability [40,44,46], resulting thus, in the reduction of the incidence of infectious complications (39 vs. 64%, p < 0.02). The decrease of the infectious complications (4 vs. 12 cases, p < 0.005) could be possibly influenced by the increase of the nitrogen balance (12 ± 2 vs. 5 ± 1 g N, p < 0.05), as well as the levels of IgA (335.7 ± 31.44 vs. 357.81 ± 83.61 mg/dL, p = 0.029) and IgG between intervention and the control group [81].
Sahin et al. [88] reported a lessening in CRP in both groups, although the decrease was more pronounced in the study group (−38 vs. 18.6%, p = 0.00 and p = 0.01, respectively). However, these values were still higher than usual and a reason for this could be the presence of fewer leukocytes, and inflammation. In this trial, there was a reduction in LOS (14.2 ± 4.4 vs. 16.4 ± 3.9 days, p > 0.05) and complication rates (10 vs. 40%, p < 0.05) which may be due to an increase in CD4 and CD8 lymphocytes, although the latter failed to reach significance (p > 0.05). The increase in T-cell lymphocytes was also reported in other studies [80,83,87]. Nevertheless, in line with the latter, these measurements did not reach significance (p > 0.05).
According to a review conducted by Novak et al. [54], the minimum concentration of glutamine to obtain positive clinical outcomes is 0.2 g/kg/day Gln, corresponding to 0.303 g/kg/day Ala-Gln dipeptide. Nevertheless, this amount could be raised to 20 g/day Gln according to Wischmeyer et al. [78], Heyland et al. [109] and García-De-Lorenzo [110]. Regarding Gln toxicity, Garlick [111] suggested that doses as high as 50-60 g/day Gln for several weeks were safe and showed no adverse effect. The use of a lower amount of glutamine could in part explain the lack of significance in the reduction of cytokines levels in some studies [69]. In the study by Engel et al. [63], dose requirements according to the previous studies [54,78,109,110] were met. In this case, a reason to explain these results may be the short time used for supplementation (3 days) rather than the dose used.
The form of supplementation used might also affect the outcome, even if the minimum amount of glutamine is given. This could be explained since glutamine in the form of L-Gln presents much lower stability than Ala-Gln dipeptide [78]. In addition, the effect of glutamine may also vary depending on whether it is given postoperatively and parenterally, and whether it is given preoperatively [95,104] and enterally [103], respectively. The observed beneficial effect on L/M ratio and TNFα levels was more pronounced when glutamine was administered by the enteral route. However, since the authors do not specify the amount of the administered glutamine, it is difficult to determine whether this observation is dose dependent or is due to the method of administration being the enteral route.
The shortest study [63] supplemented patients for just 3 days with 0.5 g/kg/day Gln in a double-blind RCT. In this trial, no significant differences were found in total lymphocytes count (p > 0.05), IL-6 levels (p < 0.05), LOS (2.6 ± 2.0 vs. 2.0 ± 1.7, p = 0.44), CRP (p = 0.72), IL-8 (p > 0.05) or TNFα (p > 0.05). This might suggest that a minimum length of 5 days is needed to achieve an effect in at least one of the parameters related to healing [69].
Besides, according to Morlion et al. [71], glutamine supplementation may also have a positive effect on the patient's mood and general well-being. A reason for this could be the role of Gln as a neurotransmitter [112,113] as its depletion has been shown to cause mood disorders such as depression [113][114][115].
Therefore, it would be useful to measure this parameter in future studies to corroborate this effect.

Limitations of the Review
In the case of arginine, five studies were included in the meta-analysis. However, it was not possible to assess the effect of arginine supplementation on nitrogen balance and T-cell lymphocytes due to the lack of studies reporting these data. Besides, the studies had relatively small sample sizes. Therefore, more studies are required in this area of research. Besides, the high heterogeneity of the studies of both amino acids might have also affected the results of the meta-analysis.

Conclusions
This systematic review and meta-analysis have demonstrated that supplementation with either arginine and glutamine can positively influence wound healing or parameters related to healing including LOS and mortality. The effect of arginine supplementation was significant in relation to hydroxyproline content (p < 0.00001), while glutamine supplementation had significant effect on nitrogen balance (p < 0.0001), patient mortality (p = 0.0004), L/M ratio (p < 0.00001), LOS (p < 0.00001), CRP (p < 0.00001), IL-6 levels (p = 0.0001) and TNFα levels (p < 0.00001). However, the effect of glutamine supplementation on T-cell lymphocytes failed to reach significance (p = 0.07).