Abstract

Background: This study aims to determine the effect of glutamine-enforced parenteral nutrition on immune function and stress response of patients after esophageal operation.

Methods: A total of 143 esophageal cancer patients (71 males, 72 females; mean age 70.7±4.3 years; range 35 to 80 years) undergoing the same elective operation were randomized into two groups as the experimental group which was given additional alanyl-glutamine (0.35 g/kg/day glutamine) and the control group which was performed simple vein injection. All patients were infused with equal calorie and nitrogen by central vein during the postoperative seven days. Patients’ immunological and inflammatory parameters were analyzed.

Results: Preoperatively, the levels of immunoglobulin A, G, M levels, CD3+ and CD4+ T lymphocytes, and CD4+/CD8+ T lymphocytes ratio of both groups were lower than normal values. The levels of complement 3 (C3), C4 and C reactive protein of both groups were higher than normal values. On the first postoperative day, all indicators of experimental group were superior to the control group except for levels of C reactive protein, immunoglobulin M, and CD3+ and CD8+ cells (p>0.05). On the seventh postoperative day, the C reactive protein level of experimental group was lower than that of control group (p<0.05), while the CD4+ cells, immunoglobulin A and G levels of experimental group were higher than the control group (p<0.05).

Conclusion: Compared with conventional parenteral nutrition, glutamine-enforced parenteral nutrition support may significantly improve the immune function and reduce stress response in patients after esophageal operation.

Esophageal cancer is not an infrequent malignant tumors in China. Worldwide, almost 400,000 new cases of esophageal cancer are diagnosed annuallyit is the eighth most common cancer and the sixth most common cause of cancer-related mortality.[1] China carries a big burden of esophageal cancer with an incidence of 19.3 per 100,000 in 2008,[2] and accounts for more than half of the new cases diagnosed in the world.[3] Due to tumor and intestinal obstruction, these patients are affected by negative nitrogen balance, stress, and the suppression of the immune function to some degree. Operation is the main treatment. However, operation trauma and stress aggravate the condition. Studies have shown that a weakened immune system is the main cause of infection and even death after operation.[4] Furthermore, conventional nutritional support may not improve the immune function of patients and reduce stress reaction. Until today, the commonly studied immune nutrients are glutamine (Gln), arginine, and omega-3 fatty acids.[5] According to a clinical study, adequate supplementation of Gln may improve immune response under stress, shorten recovery time, and reduce mortality rate.[6] Therefore, in this study, we aimed to determine the effect of Gln-enforced parenteral nutrition on immune function and stress response of patients after esophageal operation.

Methods

The study included a total of 143 esophageal cancer patients (71 males, 72 females; mean age 70.7±4.3 years; range 35 to 80 years) who admitted to the Affiliated Hospital of Putian University between August 2009 and October 2013. Distribution of esophageal cancer types was as follows: upper thoracic esophageal cancer in 22 patients, middle thoracic esophageal cancer in 83 patients, and lower thoracic esophageal cancer in 38 patients. All patients were randomized into two groups as the experimental group and control group by means of New Drug Statistical Treatment. Patients in both groups had no significant differences in terms of sex, age, tumor location, operation methods of anastomosis, pathological stages, time of the beginning of preoperative symptoms, preoperative nourishment status, preoperative immune status, and stress reaction (Table 1).

Table 1: Clinical parameters of patients in two groups

Inclusion criteria: (i) operation and pathology reports for esophageal squamous cell carcinoma, negative margin; (ii) n o i ndication o f m etabolic diseases such as diabetes or hyperthyroidism; (iii) no hematopoietic system disease; (iv) no spleen and thymus resection; (v) no autoimmune disease with negative effect on immune function; (vi) no exposure to chemotherapy, and hormone or immune stimulators.

Excluded patients: (i) patients undergoing operation difficulty; (ii) patients receiving more than 800 mL of blood transfusions; (iii) p atients r eceiving i nfusion o f albumin during operation.

Experimental and control groups had isonitrogenous and isocaloric intake of nutrition from the first postoperative day for seven days. The nutrition given continuously for seven days included 30 kcal/kg/day; glucose, 6:4 ratio; nitrogen, 0.3 g/kg/day; heat, 100 kcal:1g nitrogen. The experimental group was given additional alanyl-Gln, according to 0.5 g/kg/day (Gln 0.35 g/kg/day) supply.

On first preoperative day, and on first and seventh postoperative days, the following parameters were measured: concentrations of Gln in serum; stress response indicators: serum C-reactive protein (CRP), complement 3 (C3), C4; humoral immunity indexes: serum immunoglobulin (Ig)M, IgA, IgG; and lymphocyte-mediated immune parameters: CD3+, CD4+ and CD8+ lymphocytes.

The experimental protocol of the study was approved by Department of Thoracic Surgery in the Department of Thoracic Surgery in the Affiliated Hospital of Putian University. A written informed consent was obtained from each patient. The study was conducted in accordance with the principles of the Declaration of Helsinki.

Statistical analysis
All data were analyzed using SPSS for Windows version 16.0 software (SPSS Inc., Chicago, IL, USA). Student’s t test was used for the measurement data such as age, stress response indicators, and immunity indexes, expressed as mean ± standard deviation. Chi square test was used for the enumeration data such as tumor location and mode of operation. P value <0.05 was considered statistically significant.

Results

On preoperative first day, levels of CRP, C3, and C4 of both groups were higher than normal, but the comparison between two groups showed no significant difference. On postoperative first day, levels of C3 and C4 of the experimental group were lower than the control group (p>0.05), while CRP level of the experimental group was higher than the control group (p>0.05). On seventh postoperative day, CRP level of the experimental group was significantly lower than that of the control group (p<0.05), (Table 2).

Table 2: Comparison of inflammatory parameters before and after operation

On preoperative first day, levels of IgA, IgG, IgM, CD3+ and CD4+ cells, and CD4/CD8 ratio of both groups were lower than normal, but the comparison between two groups revealed no significant difference. On postoperative first day, levels of IgA and IgG of the experimental group were higher than the control group (p>0.05), while IgM of the experimental group was lower than the control group (p>0.05). Levels of CD4+ and CD8+ cells, and CD4/CD8 ratio of the experimental group were higher than those of the control group (p>0.05), while CD3+ cells of the experimental group was lower than the control group (p>0.05). On postoperative seventh day, levels of CD3+ cells, CD4/CD8 ratio, and IgM level of the experimental group were higher than those of the control group with no statistical significance (p>0.05), level of CD8 of the experimental group was lower than the control group with no statistical significance (p>0.05), while levels of CD4+ cells, IgA, and IgG of the experimental group were significantly higher than those of the control group (p<0.05), (Table 3, 4).

Table 3: Comparison of humoral immune parameters before and after operation

Table 4. Comparison of cellular immune parameters before and after operation

On preoperative first day, the concentrations of Gln in serum of both groups were normal. On postoperative first day, the concentrations of Gln of both groups were lower than those of first preoperative day in each group (p<0.05). The concentrations of Gln of the experimental group were higher than the control group (p>0.05). On seventh postoperative day, the concentrations of Gln of the experimental group were higher than that of the control group (p<0.05). The concentrations of Gln of the control group were lower than those of first preoperative day (p<0.05), (Table 5).

Table 5: Comparison of the concentrations of glutamine in serum before and after operation (μmol/L)

The incidence of pulmonary infection and anastomotic leakage of the experimental group was lower than that of the control group (p<0.05). The incidences of incision infection, blood infection, urinary infection, and abdominal infection of the experimental group were lower than that of the control group (p>0.05), (Table 6).

Table 6: Comparison of the infectious complication between two groups

Discussion

Esophageal cancer is one of the least studied and most lethal cancers worldwide,[7-9] with a five-year survival rate of less than 10%.[10] It is one of the most common malignant tumor in China. Its incidence has risen in recent years.[11] Patients usually have various degrees of malnutrition, even the emergence of cachexia due to patients’ difficulty in eating and the metabolic disorder of the organism because of increased production of cytokines and catabolic hormone.[5] At the same time, preoperatively, esophageal cancer often causes low immune function and stress response in different degrees. These also may increase the risk of infection and tumor recurrence, and affect prognosis. Therefore, improving the nutritional status and recovering the immune function of patients postoperatively are important to improve their stress response.

Glutamine is a nonessential amino acid, which is an important energy metabolism of immune cells and intestinal mucosal cells. It can also promote the division and differentiation of lymphocytes and macrophages. It plays a significant role in human nutrition metabolism, immune protection, and regulation of inflammation.[12] When the body is under stressful or pathological conditions, when there is a relative lack of endogenous synthesis of Gln, or in case of Gln depletion, exogenous Gln must be supplied. Under these conditions, Gln becomes an essential amino acid. Therefore, Gln is considered to be a conditionally essential amino acid for the body in case of stress, which is important for cell metabolism and immune cell function. In our study, the concentrations of Gln in serum of both groups were normal preoperatively. Postoperatively, esophageal cancer patients have continuous high rates of metabolism decomposition, while Gln consumption and blood Gln utilization rate increase. Similarly, on first postoperative day in our study, the concentrations of GLN of both groups were lower than those of the first preoperative day in each group. Insufficient nutrition supply may induce intestinal mucosal atrophy, which aggravates stress reaction of metabolism. In their study, Zhou et al.[13] demonstrated Gln as an additional nutrition for some severely burned rats and that it can reduce intestinal mucosal reaction of metabolism. Therefore, in traumatic stress, percutaneous enteral or parenteral Gln nutrition support might maintain the structure and function of intestinal mucosa and reduce skeletal muscle protein degradation, improve nitrogen balance, and reduce the stress reaction of metabolism. In our study, when the body’s immunological function gradually restored, the concentrations of Gln in serum of experimental group became normal. However, on the seventh postoperative day, the concentrations of Gln in serum of control group were lower than the experimental group because of the relative lack of endogenous synthesis of Gln, or even body Gln depletion. In addition, CRP is a sensitive index, which reflects stress status, while C3 and C4 synthesis increases the condition of stress.[14] Our study confirmed these results. Preoperative CRP levels in both groups were higher than normal values, suggesting that patients were under the state of stress response before operation. On the first postoperative day, CRP levels of the two groups were further increased, indicating that operation trauma aggravates stress response even more; however, on the seventh postoperative day, CRP level of the experimental group significantly decreased, while that of the control group showed no obvious decrease. There was a significant difference between the two groups (p<0.05), suggesting that Gln-enforced parenteral nutrition might ameliorate stress response in patients with esophageal cancer postoperatively.

On the first preoperative day, levels of IgA, IgG, and IgM of both groups were lower than normal, suggesting that patients were under the protection of humoral immunity suppression. On the seventh postoperative day, levels of IgA and IgG of the experimental group were significantly higher than the control group (p<0.05), indicating that Gln-enforced parenteral nutrition might enhance humoral immunity of patients with esophageal cancer postoperatively. It may be so since Gln is the precursor to purine, pyrimidine, nucleic acid, and protein. Also, it is a carrier of nitrogen between tissues and organs. Moreover, Gln plays an important role in promoting B lymphocyte synthesis and secretion of antibodies, and this effect cannot be replaced by other kinds of amino acid.[15]

On the first preoperative day, the levels of CD3, CD4, and CD4/CD8 of both groups were lower than normal, suggesting that patients were under the protection of cell-mediated immunity suppression. On the seventh postoperative day, CD4 level of the experimental group was significantly higher than the control group (p<0.05), indicating that Glnenforced parenteral nutrition might enhance cell immunity in patients with esophageal carcinoma postoperatively. It may be so since Gln is the precursor to synthesis metabolism, given the fact that the need for Gln increases in the condition of stress. Glutamine supplementation can improve the protein metabolism, thereby inhibiting muscle protein breakdown, improving nitrogen balance, and promoting gluconeogenesis. At the same time, Gln is capable of improving the physiological function of immunologically competent cells, such as intestinal mucosal cells and lymphocytes, macrophages and neutrophils.[11] Meanwhile, whether in enteral or parenteral application, Gln is equally effective.[16] Therefore, the incidence of pulmonary infection and anastomotic leakage of the experimental group was lower than the control group in our study (p<0.05).

This study has some limitations. The outcome is only in the form of biochemical surrogate markers. No information is given on postoperative mortality between the two groups. And this is essential information since it is a major outcome. Furthermore, in this case, although the study of parenteral glutamine could be useful enough for proof of principle, enteral administration may have more clinical relevance. These questions should be substantially evaluated in future studies.

In conclusion, this study showed that patients with esophageal cancer are affected by decreased immune function and high stress postoperatively. Compared with conventional parenteral nutrition, glutamineenforced parenteral nutrition support may improve the immune status, reduce stress response significantly, and thereby promote recovery.

Acknowledgment
We thank Chen Si-Zeng (Department of Gastrointestinal Surgery, the First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian, China) for his assistance in editing this manuscript.

Declaration of conflicting interests
The authors declared no conflicts of interest with respect to the authorship and/or publication of this article.

Funding
The authors received no financial support for the research and/or authorship of this article.

References

1) Wan QL, Hou XS, Zhao G. Utility of serum peptidome patterns of esophageal squamous cell carcinoma patients for comprehensive treatment. Asian Pac J Cancer Prev 2013;14:2919-23.

2) Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer 2010;127:2893-917.

3) Zhao J, He YT, Zheng RS, Zhang SW, Chen WQ. Analysis of esophageal cancer time trends in China, 1989- 2008. Asian Pac J Cancer Prev 2012;13:4613-7.

4) Hotchkiss RS, Karl IE. The pathophysiology and treatment of sepsis. N Engl J Med 2003;348:138-50.

5) Li JS. Nutritional oncology and it's clinical application. Parenteral and Enteral Nutrition 2004;11:1-2.

6) Aosasa S, Mochizuki H, Yamamoto T, Ono S, Ichikura T. A clinical study of the effectiveness of oral glutamine supplementation during total parenteral nutrition: influence on mesenteric mononuclear cells. JPEN J Parenter Enteral Nutr 1999;23:41-4.

7) Lin JY, Wang MS, Dong LP, Xu DW, Wang Y, Li XL, et al. Influence of personal character on quality of life of patients with esophageal cancer in north Henan province and influencing factors. Asian Pac J Cancer Prev 2012;13:5415-20.

8) Lin JY, Qin JB, Li XY, Dong P, Yin BD. Diagnostic value of human epididymis protein 4 compared with mesothelin for ovarian cancer: a systematic review and meta-analysis. Asian Pac J Cancer Prev 2012;13:5427-32.

9) Ma JB, Chen EC, Song YP, Liu P, Jiang W, Li MH, et al. Prognostic significance of 18F-fluorodeoxyglucose positron emission tomography (PET)-based parameters in neoadjuvant chemoradiation treatment of esophageal carcinoma. Asian Pac J Cancer Prev 2013;14:2477-81.

10) Lee JM, Wu MT, Lee YC, Yang SY, Chen JS, Hsu HH, et al. Association of GSTP1 polymorphism and survival for esophageal cancer. Clin Cancer Res 2005;11:4749-53.

11) Xia HH, Zhang ST, Lam SK, Lin MC, Kung HF, Wong BC. Expression of macrophage migration inhibitory factor in esophageal squamous cell carcinoma and effects of bile acids and NSAIDs. Carcinogenesis 2005;26:11-5.

12) Yang R, Tan X, Thomas AM, Steppacher R, Qureshi N, Morrison DC, et al. Alanine-glutamine dipeptide (AGD) inhibits expression of inflammation-related genes in hemorrhagic shock. JPEN J Parenter Enteral Nutr 2007;31:32-6.

13) Zhou YP, Jiang ZM, Sun YH, Wang XR, Ma EL, Wilmore D. The effect of supplemental enteral glutamine on plasma levels, gut function, and outcome in severe burns: a randomized, double-blind, controlled clinical trial. JPEN J Parenter Enteral Nutr 2003;27:241-5.

14) Hossain MZ, Latif SA, Uddin MM. Is serum C-reactive protein really a sensitive stress marker in Bangladeshi people. Mymensingh Med J 2005;14:16-8.

15) Crawford J, Cohen HJ. The essential role of L-glutamine in lymphocyte differentiation in vitro. J Cell Physiol 1985;124:275-82.

16) Zhang GY, Wu CJ, Huang LM. Application of L- alanyl -L- glutamine (LALG) intensified parenteral nutrition support in critically surgical patients. Herald of Medicine 2009;28:318-20.