Intestinal intraluminal injection of glutamine increases trolox total equivalent antioxidant capacity ( TEAC ) in hepatic ischemia-reperfusion

Purpose: To evaluate the effects of intraluminal injection of glutamine on the serum trolox equivalent antioxidant capacity in an experimental model of ischemia-reperfusion of the liver observing the applicability of modifications on the original assay method. Methods: Thirty Wistar rats underwent laparotomy to perform a 20 cm blind sac of small bowel and occlusion of the hepatic hilo for 30 minutes and reperfusion for 5 minutes. Into the gut sac it was injected glutamine (glutamine group, n=10) or distilled water (control group, n=10). Ten other animals (sham group) underwent laparotomy without artery occlusion. Blood samples were collected for trolox equivalent antioxidant capacity assays in different temperature conditions, reagent quantities and time for spectrophotometer readings. Results: Total antioxidant capacity was significantly greater in glutamine group than in both control group (1,60[1,55-1,77] vs 1,44[1,27-1,53]) and sham group (1,60[1,55-1,77] vs 1,48[1,45-1,59]). Conclusion: Glutamine enhanced serum antioxidant capacity. The assay technique consistently reflected the changes in the antioxidant defenses in this experimental model.


Introduction
In various clinical and surgical conditions an ischemic event may play a crucial role in the pathophysiology of cell damage.Reperfusion to reestablish the blood flow to the tissues after ischemia is fundamental in the management of this condition.However, reperfusion may be responsible for injuries for the whole organism that are most serious than the ischemic damage per se 1 .The conjunction of the pathophysiologic events and the severe clinical condition that follows is called ischemia and reperfusion injury (IRI).In modern hepatic surgery prevention of IRI is most important and relevant.Pringle 2 in 1908 described the classical operative procedure to clamp the portal triad to control intra-operative hemorrhage.This procedure is currently used in trauma surgery and during resection of the liver 3 .Orthoptic liver transplantation may constitute a major prototype and good example of IRI in the liver.The formation of oxygen free radical in the ischemic tissues may play an important role in IRI 4 .Anti-oxidative natural defenses are innocuous to limit the huge formation of free radical that follows and thus, oxidative damage is eminent 5 .Among the various anti-oxidative systems it is thought that glutathione system is the major biological mechanism to antagonize the creation of these oxygen free radicals 6 .Glutamine is the most abundant amino acid in the plasma .Glutamine is the principal fuel for the enterocytes and immune cells, and is avidly taken by the intestinal epithelium cell by both the lumen and blood poles 8 .Glutamine is fundamental for glutathione formation 9 .The liver is the most important reservoir of glutathione, which is composed of three amino acids connected in tandem: glycine, cysteine, and glutamate, that is derived from glutamine 6 .Aguilar-Nascimento et al 10 showed that intraluminal injection of glutamine minimize intestinal injury and the afflux of neutrophils after small bowel IRI.They suggest the findings are probably due to the increase of the anti-oxidative defenses by the presence of glutamine.The search for substances with anti-oxidative properties are a challenge for investigators.Methods to evaluate the anti-oxidative status are most important in IRI because they can be use in many diseases and conditions 11,12 .However, it is very difficult to evaluate each component of the complex antioxidative system and their interactions 13 .Thus, various methods have been reported to measure the total antioxidative capacity of the serum 14,15,16,17,18 .However, most of them are time-consuming and expansive.Miller et al 17 reported a colorimetric method for the evaluation of the anti-oxidative capacity of the serum that was posteriorly modified by Re et al 19 .This technique is based on the inhibition of the absorbance of the free-radical ABTS *+ by anti-oxidants, and on the relation of this radical cation with the anti-oxidant scavenger Trolox, which is the synthetic analogue of Vitamin E. This method is very easy to be performed and most accurate 20 .In this present study we aimed at evaluating the effects of intraluminal glutamine in the Trolox total anti-oxidative capacity (TEAC) in an animal model of IRI of the liver.

Experimental design
Thirty male Wistar rats (250-300g) from the Central Biotery of the Federal University of Mato Grosso entered the experiment.Firstly they were kept during three days at the Investigation Laboratory of the Medical Sciences School environment at a constant temperature (25 o C) in 12/12h light-dark cycles, and receiving water and rat chow ad libitum for adaptation.All experiment follows the ethical guidelines of the Brazilian College of Animal Experimentation (COBEA).The animals underwent 5cm median laparotomy under anesthesia with inhalatory ether associated with intramuscular infection of 50 mg/kg ketamin chloride.During the operation the ileo-cecal valve was localized and at this point a 3-0 cotton suture was passed and tied by a mesenteric breach to occlude the intestine.Twenty cm up to this distal suture another similar suture for occlusion of the proximal small bowel was done and a closed 20 cm length small bowel sac was created.A careful dissection of the hepatic pedicle was performed and the vascular structures were clamped by a Pringle maneuver using an atraumatic vascular clamp 2 .At the same time, the intestinal sac was punctured by a 13x4.5mmneedle and either a 1 mL solution containing of 20% glutamine (glutamine group; n=10) or distilled water (control group; n=10) was injected inside the gut lumen.A previous pilot study attested that this volume was enough to entirely fill the sac without promoting significant distention of the gut sac.In a third group of rats (sham group, n=10) the hepatic pedicle was not clamped and nothing was injected into the small bowel sac.After 30 minutes of ischemia the vascular clamp was released and reperfusion was allowed for five minutes.Blood samples (3-4 mL) from the thoracic posterior vena cava was obtained and then the animal was killed with an overdose of inhalatory ether.In animals from the sham groups blood samples were collected after 35 minutes of anesthesia and laparotomy.Animals were allocated in the various groups at random during operation.Blood samples collected in heparinized syringes were immediately centrifuged at 1500 rpm for 20 minutes.Approximately 1.5 mL of the supernatant layer corresponding to the serum was placed in 2mL tubes, frozen and stocked in liquid nitrogen until the time of the assay.

Trolox Equivalent Antioxidant Capacity Curve
A calibration curve for the Trolox equivalent antioxidant capacity was built by plotting different concentrations of Trolox (mM) versus its total equivalent antioxidant capacity.Table 1 show the concentrations used in the curve.Approximately 2970µl of the working solution containing the radical ABTS •+ was pipetted and placed in plate of spectrophotometer.Absorbance at 734 nm was read and corresponded to T0. Thirty µl of trolox solution was added and the final solution was manually mixed for 20 seconds and then read it again at 734 nm and the value obtained after exact five minutes (T5) was registered.The assay was repeated in triplicate for each of the trolox dilution (A to F tubes).The antioxidant capacity was defined by the value of the difference between T5 and T0 divided by T0 ([T5-T0]/T0).For corrections related to the phosphate buffer the final value was lessened from the value observed in the F tube (white control).Figure 1 shows the pattern curve of the Trolox total antioxidant capacity correlated to its final concentration.The reaction is linear and of the first degree for the interval corresponded to a inhibition potential of about 40-80% 1 .

Serum total Trolox equivalent antioxidant capacity
Measurements were done in duplicate following the same protocol used for the standard curve.The total antioxidant activity (TAA) was calculated by the formula:

Statistical analysis
Data was analyzed by the Kolmogorov-Smirnov test for normality and by the Levene's test to assure homogeneity.The Kruskal-Wallis was used for comparisons between groups and the Mann-Whitney test used if any statistical significance was found.A level of 5% (p<0.05) was established as significant.Data was expressed as median and range.], p<0,05).There was no difference between sham and control groups (p= 0.10).These data can be seen in figure 2.

TABLE 1 -
Concentrations used to build the pattern curve of the total Trolox equivalent antioxidant capacity.
PBS = phosphate buffer solution