Glutathione, a tripeptide (glycyl-glutamyl-cysteine), is one of the most important antioxidants in human cells. It has many protective and metabolic functions within the cells, including the regulation of protein synthesis and many enzymatic reactions. It acts as a scavenger that protects protein thiol groups from free-radical-induced oxidant injury, especially for maintaining cellular integrity. Particularly in the liver, glutathione is also involved in detoxification and metabolism of a number of substances.1,2 Experimental and clinical research have indicated that tissue glutathione stores and plasma glutathione levels are affected during trauma or serious illnesses. In addition, glutathione has been shown to be depleted during critical illness. A glutathione-deficient state is associated with multiple-organ dysfunction and leads to marked mortality in intensive care unit (ICU) patients.1,3–7
After surgical trauma, glutathione concentrations decrease in skeletal muscle. However, IV glutamine supplementation attenuates glutathione depletion in skeletal muscle in humans after standardized surgical trauma.8 Alanyl-glutamine dipeptide IV supplementation has been shown to prevent worsening of insulin sensitivity in multiple-trauma patients.9 Total parenteral nutrition (TPN) supplemented with alanyl-glutamine dipeptide in ICU patients is associated with a reduced rate of infectious complications and better metabolic tolerance.10
The aim of this clinical study was to investigate the effect of IV alanyl-glutamine dipeptide supplementation on plasma glutathione levels in severely traumatized patients receiving standard enteral nutrition.
After the approval of our institutional ethics committee and written informed consent from the patients’ closest family members were obtained, 40 patients with severe trauma were enrolled in this study. Inclusion criteria were age from 18 to 65 yr and severe multiple injury based on an Injury Severity Score (ISS) >20. Exclusion criteria were pregnancy and coexisting disease before trauma, body mass index of >30 or <18.5 kg/m2, Glasgow score <5 for head trauma, renal replacement therapy, and a contraindication for enteral nutrition. At admission in the ICU, demographics and clinical information were obtained. The severity of anatomic injuries was calculated according to the ISS. The Acute Physiology and Chronic Health Evaluation was performed at admission, and the Sequential Organ Failure Assessment was performed every day of the study.
Forty trauma patients were randomly assigned to two groups using sealed, shuffled, numbered, and opaque envelopes. Twenty patients received IV alanyl-glutamine supplementation (Dipeptiven, Fresenius Kabi) (Group G), and 20 patients received a control solution without alanyl-glutamine (Group C) in a double-blind manner. All patients were fed the enteral commercial formula (Biosorb standard, Nutricia) (protein 16%, carbohydrate 49%, fat 35%, and standard vitamins, minerals, trace elements, and electrolytes 1 kcal/mL, osmolarite 265 mOsm/L, 500 mL) via a nasoduodenal tube. The location of the feeding tube was radiographically controlled. The enteral feedings were started within 24 h after admission with a rate of 25 mL/h and were gradually increased until the subject received the targeted nutritional intake. Energy needs were calculated according to the Harris-Benedict equation. The study solutions, Dipeptiven, 20 g N(2)-l-alanyl-l-glutamine (=8.20 g l-alanine, 13.46 g l-glutamine) in 100 mL, Fresenius Kabi, or saline (0.9% NaCl, 100 mL), were administered continuously, 0.5 g · kg−1 · d−1 using a central vein for 7 days.
All patients were treated by the same therapeutic protocol, including the use of analgesics, sedatives, close glucose monitoring, Centers for Disease Control criteria for septic complications and antimicrobial drugs, diuretics, H2 blockers, and ventilator regimes, among others. Blood samples were taken via radial artery catheter for analysis of total glutathione, C reactive protein (CRP), prealbumin, and glucose before the initiation of alanyl-glutamine or control solution supplementation and on the 3rd, 7th, and 10th days of feeding. All blood samples were sent routinely to a biochemistry laboratory for analysis of CRP, prealbumin, and glucose. For glutathione measurements, the blood samples were centrifuged and their plasmas separated, and the plasmas were stored at −20°C until they were analyzed. Glutathione analysis was performed with the Glutathione Assay Kit 703002 (Cayman Chemical Company, Ann Arbor, MI). Results, number of days on mechanical ventilation (MV), total ICU length of stay, 1-mo mortality rate, and complications were also recorded.
A power analysis was performed on the basis of the difference in glutathione levels between the alanyl-glutamine and control groups on Day 7 after the supplementation. Based on previous clinical experience, assuming a two-tailed Type 1 error of 0.05 and ß-error of 0.1, approximately 18 patients in each group were required to detect a difference of 0.20 (sd = 0.18) in the glutathione levels between the groups. Twenty patients were studied in each group; thus, the study reached a power of 90%.
After data acquisition, the arithmetic mean ± sd was calculated for each group. The Kolmogorov-Smirnov test was used for normality and homogeneity of data distribution. Continuous data (age, weight, height, ISS, Acute Physiology and Chronic Health Evaluation II, glutathione, CRP, prealbumin, and glucose) were compared between the groups using Student’s t-test. Dichotomous data (sex, complication) between the groups were compared with the χ2 test. Glutathione, CRP, prealbumin, and glucose levels were analyzed by two-way analysis of variance followed by post hoc Dunnett for repeated measurements. A value of P ≥ 0.05 was taken as statistically significant.
Admission characteristics of the patients were similar for the two groups (Table 1). Within the study period, two patients died (one in Group G on study Day 14 and one in Group C on study Day 12), and 38 patients completed the study. After alanyl-glutamine supplementation, plasma total glutathione levels significantly increased in Group G when compared with Group C on Day 7 and on Day 10 (1.34 ± 0.20 μM vs 1.13 ± 0.14 μM, and 1.38 ± 0.19 μM vs 1.12 ± 0.16 μM) (P < 0.001) (Fig. 1). There were no differences in CRP, prealbumin, and glucose levels between the two groups. No significant difference was found in Sequential Organ Failure Assessment scores on any day of the study.
The number of nosocomial infections (8 vs 10 overall, 4 vs 5 pneumonias, 1 vs 1 ventilator-associated pneumonias, 2 vs 3 bacteremias, 1 vs 1 wound infections), mean MV days (8 ± 3 vs 9 ± 3), mean length of stay in the ICU (14 ± 2 days vs 15 ± 2), and presence of metabolic disorders were not different between Group G and Group C. One patient died on study Day 12 in Group C and one on study Day 14 in Group G. The number of survivors at 1 mo was similar between the two groups.
This is the first study addressing the effect of IV dipeptide alanyl-glutamine supplementation on plasma glutathione levels in critically ill trauma patients receiving standard enteral nutrition. Plasma glutathione levels are maintained primarily by a balance between secretion from the liver and degradation in the kidney and other tissues. Hepatic glutathione depletion has been observed after hypovolemic shock, sepsis, multiple trauma, and malnutrition.1–3 Luo et al.1 demonstrated that the concentrations of the reduced glutathione and the total form of glutathione were affected in muscle tissue and blood after surgical trauma, and that the glutathione level of plasma was 20% lower after operation compared with before operation (P < 0.05). In another study, Lyons et al.11 reported that glutathione synthesis rates in whole blood were decreased by 60% in pediatric critically ill septic patients receiving suboptimal nutritional support. In addition, posttraumatic glutathione depletion can be attenuated by IV glutamine supplementation, which might decrease the susceptibility of the tissues to oxidative damage.8 Another study reported that parenteral supplementation with alanyl-glutamine at a dosage of 0.4 g (corresponding to 0.28 g of glutamine) per kilogram of body weight per day prevented worsening of insulin-mediated glucose disposal observed in the control group, offering a possible additional approach to glycemic control in multiple-trauma patients.9 In this study, when trauma patients receiving enteral standard nutrition were provided with adequate alanyl-glutamine support (0.5 g · kg−1 · d−1) for 7 days, their plasma total glutathione levels increased. The regulation of glutathione concentration involves the release of glutathione precursors from peripheral tissues, uptake of precursors into its cells, de novo synthesis of glutathione within the cells, and transportation to plasma.12 Severe trauma does not decrease plasma levels of glutathione when increased support of the amino acids related to glutathione metabolism is provided. Denno et al.13 reported that glutamine-enriched TPN enhanced plasma glutathione stores in the resting state, and they suggested that glutamine was rate limiting for glutathione biosynthesis during TPN infusion. This study did not show differences between the groups in the number of nosocomial infections, presence of metabolic disorders, mean MV days or ICU days, death during the study period, or survivors at 1 mo.
This study has some limitations related to clinical outcome. First, it has limited statistical power because of the small number of patients, and second, the mortality rate may be low because the follow-up period was limited to 1 mo. Another study with a larger number of patients may demonstrate improved clinical outcome with increased plasma glutathione levels after alanyl-glutamine supplementation.
In conclusion, this study demonstrates that IV alanyl-glutamine dipeptide supplementation for 7 days increases total plasma glutathione levels in critically ill trauma patients receiving standard enteral nutrition. The contributions of the plasma glutathione levels to clinical outcome of severe trauma patients in the ICU remain unclear and require further investigation.
The author thanks Birgul Vanizor, PhD, for her helpful analysis of glutathione, and to Fresenius Kabi for providing glutathione kits. The author also thanks the ICU staff for their support in the completion of this study.
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© 2009 International Anesthesia Research Society
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