Surgery, a major iatrogenic stress, can result in a series of metabolic complications, such as hypermetabolism, hyperglycaemia, immune suppression and increased urea production with negative nitrogen balance, which will become worse if the patient undergoes prolonged fasting and preoperative enemas for surgical purposes such as digestive tract surgery.1,2 These so-called metabolic stress responses will influence the patient's outcomes, and insulin resistance, as a marker of metabolic stress, is strongly associated with the recovery after major surgery.3,4 Radical resection is a primary therapeutic modality for patients with colorectal cancer, but produces more severe negative effects on the patient's recovery by the heightened insulin resistance. However, strategies to reduce surgical stress and prevent postoperative insulin resistance can produce a considerable reduction in adverse outcomes.5 A body of literature reports that pro-inflammatory cytokine is a driving factor of insulin resistance, suggesting that blockade of such cytokine would be beneficial to patient's outcomes.6,7
Glutamine, a conditionally essential amino acid, is the most abundant free amino acid in the intracellular and extracellular compartments, and our previous study in addition to other reports has shown that glutamine can attenuate pro-inflammatory cytokine release and protect against many forms of cellular injury through increasing the hexosamine biosynthetic pathway and inducing heat shock protein expression.8–10 Brennan et al.11 have shown that glutamine enhances glucose-stimulated insulin secretion in vitro, and that glutamine can prevent worsening of insulin sensitivity in multiple trauma patients. This effect is attributed to glutamine's therapeutic actions in the maintenance of cell function and in amino acid transamination.12 Furthermore, glutamine modulates the glucose-induced loss of maximal insulin responsiveness in adipocytes,13 which may have a negative influence on glucose kinetics and the degree of inflammation in patients undergoing laparoscopic gastric bypass surgery.14
In consideration of the dual role of glutamine in glucose metabolism and insulin sensitivity, further studies are needed to explore the potential effect of glutamine on both glucose metabolism and insulin sensitivity in different pathological states. We tested the hypothesis that preoperative and intraoperative intravenous supplementation of glutamine can improve glucose–insulin homeostasis and facilitate recovery in patients undergoing colonic cancer resection.
Materials and methods
This study was a prospective, randomised and single-centre clinical trial. Ethical approval was granted by the Ethical Committee of Southeast University Affiliated Zhongda Hospital, 87# Ding Jia Qiao Rd., Nanjing, China (Chairperson Prof Bao-an Chen) on 15 January 2011. Written informed consent was obtained from each of the 60 eligible patients who underwent elective colon cancer resection from January 2011 to May 2011. Inclusion criteria were patients of either sex; aged from 35 to 75 years; BMI between 18.5 and 25 kg m−2; ASA physical status I–II; diagnosed with colon cancer; and listed for elective cancer resection. Patients were excluded if they had diabetes mellitus, preoperative jaundice, or immunologic, metabolic disease, cardiovascular or cerebrovascular disease; if they were obese, had infection or critical illness; if they were unable to take oral fluids or had malabsorption of the gut; if they were using thyroid medication, corticosteroids or diuretic medication; if they were pregnant; or if surgery lasted more than 5 h, or the transfusion volume was more than 1500 ml before T3, or blood loss more than 500 ml, or they needed a blood transfusion.
A total of 60 patients were assigned to one of the three groups (20 cases each group) using a random number table by a nurse who was not involved in the study and the assignments were not revealed until 24 h before surgery. The study solutions (physiological saline, dilution vehicle or glutamine) were prepared in an unlabelled vial by an independent ward nurse following randomisation. The patients in the control group received an intravenous infusion of physiological saline; the patients in the dilution vehicle group received an intravenous infusion of 8.5% 18AA-II (an 8.5% compound amino acid injection containing 18 amino acids with a total of amino acids 85 g; Sino-Swed Pharmaceutical Corp. Ltd, Wuxi, Jiangsu, China); and patients in the glutamine group received an intravenous infusion of 3.4% alanyl-L- glutamine (Sino-Swed Pharmaceutical Corp. Ltd). The total volume of each solution was 22.4 ml kg−1 and it was administered 24 h before and 1 h after the start of surgery. The stock solution of glutamine (20%) was diluted to 3.4% with 8.5% 18AA-II for intravenous infusion. The 22.4 ml kg−1 of 3.4% Ala-glutamine provided 0.5 g kg−1 of glutamine.
Given the close relationship between insulin resistance and insulin sensitivity and that insulin sensitivity index is indicated through use of a euglycaemic hyperinsulinaemic clamp,15,16 we chose the homeostasis model assessment (HOMA-IR) and quantitative insulin sensitivity check index (QUICKI) as markers of insulin resistance and insulin sensitivity, respectively. The formulas for insulin resistance and insulin sensitivity calculations were as follows:
Anaesthetic and operative procedures
No glucose, insulin or fat emulsion was infused from 6 h before surgery until the end of surgery. All patients received intramuscular 0.1 g of phenobarbital sodium and 0.5 mg of atropine as premedication. Anaesthesia was induced with midazolam (0.05 mg kg−1), fentanyl (4 μg kg−1) and propofol (1.0 mg kg−1). Neuromuscular blockade was provided by vecuronium (0.1 mg kg−1). Anaesthesia was maintained with sevoflurane 1 to 1.5% in oxygen and infusion of propofol (4 mg kg−1 h−1), remifentanil (0.2 μg kg−1 min−1) and atracurium (0.4 mg kg−1 h−1). The depth of anaesthesia was adjusted to maintain the auditory evoked potential index (AEPI) less than 40. Mechanical ventilation was adjusted to maintain an end-tidal carbon dioxide partial pressure of 4.0 to 5.3 kPa. Nasopharyngeal temperature, mean arterial blood pressure (MAP), heart rate (HR) and pulse oximetry (SpO2) were maintained and adjusted to the normal ranges.
The total operation time, amount of intraoperative transfusion and blood loss were recorded. The time of first passage of wind after surgery and the length of hospital stay were recorded.
Blood sample analyses
Blood samples from all enrolled patients were taken at 24 h before surgery (T1), 30 min before induction of anaesthesia (T2), 2.5 h after the beginning of surgery (T3), and 1 h (T4) and 24 h (T5) after the end of surgery, for detection of serum concentrations of glucose, insulin, tumour necrosis factor-alpha (TNF-α) and free fatty acid under fasting state. Samples were centrifuged for 15 min at 4000g and 4°C, and the supernatant was collected. Glucose was measured in 5 μl serum using a Hitachi 7600 Automatic Biochemistry Analyzer (Hitachi, Tokyobaraki, Japan). Insulin was measured in 50 μl serum using the full-automatic electrochemiluminescence analyser (Roche Diagnostics, Mannheim, Germany). TNF-α and free fatty acid were analysed using the ELISA kits (R&D Systems CO. Ltd., Minneapolis, USA and Abnova CO. Ltd., Taipei, Taiwan, respectively).
Sample size calculation and statistical analyses
The sample size calculation was based on a prestudy observation in which HOMA-IR and QUICKI were the primary endpoints measured 2.5 h after the beginning of the surgery. In this pilot study, the mean differences between the groups of 18AA-II and Gln were 1.1 (SD 1.2) and 0.33 (SD 0.34), respectively. When the two-sided α was set at 0.05, and the power was set at 0.80, the minimum sample size of 19 and 17 individuals per group was needed for HOMA-IR and QUICKI, respectively. We increased the sample size by 5% to account for potential missing data and drop-outs, and set the sample size at 20 patients in each group.
All analyses were performed using the SPSS software v18 (SPSS Inc., Chicago, Illinois, USA). Categorical variables were evaluated using chi-square test. Continuous variables were presented as means and SD. Statistical analyses were carried out by paired-sample t-test in each group and one-way analysis of variance (ANOVA) or two-way ANOVA for the multiple variables at different time points among the three groups. All ANOVA tests were followed by a Bonferoni post hoc test. The null hypothesis was rejected when the P value was less than 0.05.
A total of 64 patients with colon cancer were recruited and four declined to participate in the study; 60 patients were randomised. The surgical time for all patients was less than 5 h, no patient required a transfusion over 1500 ml before T3 and no patients were excluded for other reasons or dropped-out during the study period (Fig. 1). There were no unexpected side effects. The baseline demographic and characteristic values are summarised in Table 1 and were comparable among the three groups.
The changes in blood glucose and insulin are summarised in Table 2. Surgery induced considerable increases in both blood glucose and insulin, but glutamine suppressed such changes. The values of both HOMA-IR and QUICKI were significantly changed from time T3 to time T5 in the physiological saline and 18AA-II groups (Figs 2 and 3). However, the increase in HOMA-IR and the decrease in QUICKI induced by surgery were not observed in the glutamine-treated patients.
The values of serum TNF-α and free fatty acid are summarised in Table 3. Surgery induced an increase in TNF-α but a decrease in free fatty acid. The administration of glutamine blocked these changes in TNF-α and free fatty acid. The time to the first passage of wind and the length of hospital stay were reduced in the glutamine group compared with the other groups (Table 4).
We have provided evidence that intravenous supplementation of aAla-glutamine improves patients’ overall outcomes after colon cancer resection through balancing glucose–insulin homeostasis and attenuating the release of both TNF-α and free fatty acid. Our results are concordant with other reports in which postoperative morbidity and mortality were improved after perioperative metabolic conditioning using glutamine.17 These findings demonstrate that glutamine is a preferential substrate that benefits, at least in part, patients who are at a risk of malnutrition, bacterial translocation and acquired infections after major surgeries.
The role of glutamine in glucose metabolism and insulin sensitivity has been being studied extensively in critically ill patients. Glutamine can prevent, or treat, multiple organ dysfunction syndrome (MODS) after sepsis and subsequently reduce mortality.18 Similarly, glutamine can produce a protective effect in patients undergoing different types of surgery. For instance, in studies of gastrointestinal surgery, glutamine produced a beneficial effect on patient recovery by improving nutritional status and immune function and reducing infection.19–21 Preoperative glutamine improved glycaemic control in patients with coronary artery occlusion, and reduced myocardial injury and clinical complications after cardiopulmonary bypass indicates that glutamine exerts a cardiac protective role.22,23 Although no robust evidence supports glutamine's effect on patient's recovery from cerebral surgery, laboratory preconditioning with L-Ala-glutamine in gerbils submitted to cerebral ischaemia/reperfusion injury reduces oxidative stress and degeneration of neurons in the cerebral tissue, suggesting that glutamine may benefit patients by protecting them from neuronal injury when cerebral surgery is undertaken.24 For liver transplant patients, a recent Cochrane systemic review did not find convincing benefits from nutritional interventions, including glutamine, in posttransplant morbidity, as the included studies did not assess essential clinical outcomes and were not powered for the analysis or the observed variables were not regarded as the primary endpoints.25 Our study has the same issue in that we considered HOMA-IR and QUICKI as the primary outcomes, and only the short-term hospitalisation and first passage of wind were recorded, but long-term outcomes such as mortality, recovery from cancer resection and life quality were not evaluated. These should be assessed in future studies
One conventional view considers insulin resistance as a protective response to starvation and trauma that can guarantee glucose supply to essential organs.26 However, recent findings indicate that surgery-associated severe insulin resistance makes the energy supply change from glucose to lipid, and that this response is strongly related to an increase in perioperative complications.27 HOMA-IR and QUICKI are sensitive measurements of insulin resistance and sensitivity, respectively, and can predict the overall glucose–insulin homeostasis. We chose these two variables as our primary outcomes and found that glutamine can stabilise both, suggesting that glutamine supplementation is an important manoeuvre into perioperative care.
As reviewed by Alpers,28 previously reported studies have used intravenous glutamine in doses ranging from 0.03 to 0.57 g kg−1, and with different durations of glutamine administration (between 2 and 12 days) for patients undergoing surgery or in patients with burns. All these different regimens provided robust evidence for glutamine's perioperative administration. In the present study, we used 0.5 g kg−1 of glutamine intravenously for pre and intraoperatively, and such a strategy produced beneficial results consistent with findings of other studies. We suggest that the use of preoperative and intraoperative glutamine 0.5 g kg−1 is an effective means of maintaining glucose–insulin homeostasis in patients with colon cancer.
A previous study showed that both TNF-α and free fatty acid modulate glucose transport and lipid metabolism and correlate with the increase in insulin resistance.29 A large body of evidence from animal studies shows that glutamine modulates immune and inflammatory responses by deactivating p38 mitogen-activated protein kinase (MAPK) and cytosolic phospholipase A2 via an induction of MAPK phosphatase-1, and by mediating nuclear factor-κ B negatively, which determines the expression of inflammatory cytokines.30–32 In addition, Hou et al.33 found that glutamine can enhance the recovery of colon mucosa in dextran sulphate sodium-induced colitis during which Th-associated cytokine expression, colon-inflammatory mediator production and leukocyte infiltration into tissues were suppressed by glutamine.34 Our findings in addition to the data of Lu et al.35 demonstrate that glutamine may be beneficial postoperatively to gastrointestinal cancer patients by alleviating the inflammatory responses and decreasing infectious morbidity through blocking TNF-α production. Furthermore, TNF-α has been shown to reduce insulin signalling by decreasing insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation, phosphatidylinositol 3-kinase (PI3K) and AKT activity,29 and the elevated circulating free fatty acid results in an increase in intracellular fatty acyl CoA and diacylglycerol concentrations, which finally lead to activation of PKC-θ and then IRS-1 phosphorylation.36 Our study has shown that glutamine reduced TNF-α and free fatty acid contents, suggesting that surgery-induced insulin resistance and imbalance of glucose–insulin homeostasis can be attenuated by perioperative administration of glutamine.
A recent analysis of the efficacy and cost-effectiveness of supplemental glutamine in total parenteral nutrition therapy for critically ill patients showed that Ala-glutamine improved clinical outcomes such as reduction in mortality and infection rate, and shortened ICU hospital lengths of stay, which in turn produced a concurrent saving in hospital costs.37 Our data have also shown that glutamine supplementation in colon cancer surgical patients can shorten the time to the passage of wind, indicating that it can promote functional recovery and shorten the hospital length of stay, both of which can reduce postoperative care costs by 10 to 20%. We did not observe any long-term effects of glutamine on morbidity and mortality of patients, so we are unable to determine whether glutamine supplementation had an effect on our patient's long-term outcomes.
There are several limitations to this study. One major drawback of our trial is that we did not measure the level of plasma glutamine after the different interventions. Second, although postoperative pain is a critical factor that retards patients’ recovery and hospital discharge, especially when undergoing colorectal surgery,38 we did not assess pain extensively, and we cannot exclude an influence of postoperative pain on our patients’ outcomes.
In conclusion, a growing number of reports have shown that parenteral administration of dipeptide Ala-glutamine is associated with improvements in the clinical status of patients experiencing surgery-induced stress and critical illnesses. We have demonstrated that pre as well as intraoperative intravenous Ala-glutamine can improve the functional rehabilitation of gastrointestinal tract, shorten the hospital stay and attenuate insulin resistance through reducing release of TNF-α and free fatty acid. Thus, perioperative supplementation of Ala-glutamine may offer an additional approach to reduce perioperative complications and improve outcomes of patients undergoing selective gastrointestinal tract tumour resection.
Acknowledgements relating to this article
Assistance with the study: the authors would like to thank the nurses of the Department of the Second General Surgery for their support.
Financial support and sponsorship: this work was supported by the Department of Anesthesiology, Southeast University Affiliated Zhognda Hospital, Nanjing, China.
Conflicts of interest: none.
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