Sergio Botelho Guimarães

l-Alanyl-Glutamine Preoperative Infusion in Patients with Critical Limb Ischemia Subjected to Distal Revascularization Reduces Tissue Damage and Protects from Oxidative Stress

BACKGROUND Critical limb ischemia (CLI) is the most severe form of peripheral vascular disease where there is inadequate blood flow to a limb. Our aim was to examine the effects of preoperative infusion of l-alanyl-glutamine (l-Ala-Gln) during the ischemic period and during the first 30 minutes following blood reflow in patients with CLI who are undergoing distal femoral artery bypass surgery. METHODS Thirty-two patients with CLI were alternately allocated to group 1 (saline) or group 2 (l-Ala-Gln). Saline (1000 mL) or L-Ala-Gln 250 mL plus 750 mL of saline were infused intravenously over a 3-hour period prior to surgery. Samples (muscle and blood) were collected at the beginning of the surgical procedure, at the end of ischemia, and at 15 and 30 minutes after reperfusion. RESULTS l-Ala-Gln induced elevation in glutathione (GSH) muscle concentrations while promoting reduction in thiobarbituric acid reactive substance concentrations, demonstrating enhancement of antioxidant capacity and protection from lipid peroxidation. Decreases in LDH, lactate, and glucose blood concentrations in l-Ala-Gln-treated patients suggest increased glucose utilization by muscle and peripheral tissues. Reduction in creatine phosphokinase blood concentrations may reflect smaller muscle cell damage in l-Ala-Gln-treated patients. CONCLUSION l-Ala-Gln pretreatment reduces muscle cell damage and enhances antioxidant capacity in patients with CLI.

Oxidative stress induced by torsion of the spermatic cord in young rats

PURPOSE To evaluate the effects of the oxidative stress in an experimental model of torsion/detorsion of the spermatic cord and the legitimacy of this model for oxidative stress studies. METHODS Forty-eight male Wistar rats were randomized in two groups (n=24): G-1 (Sham) and G-2 (Ischemia/Reperfusion). All rats received intraperitoneal saline injections (2.0 ml), at 21, 9, and 1 h before right spermatic cord torsion or first sham operation. Detorsion or second sham operation was carried out 3 h later followed by testis and blood samples collection (T-0). Additional samples were collected at 1-3-6 h time-points for assessment of testis malonaldehyde, glutathione, and plasma total antioxidant power (TAP). RESULTS Spermatic cord torsion/detorsion induced a significant increase in testicular malonaldehyde contents and a significant decrease in glutathione concentrations in ischemic rats compared with sham animals. Additional increase in malonaldehyde levels occurred during reperfusion in G-2 rats. TAP was similar in both groups denoting absence of systemic effects in this study. CONCLUSION Torsion/detorsion of the spermatic cord for 3 h induces significant lipid peroxidation and reduction in glutathione content of the testis and is, therefore, a valid model for studying the oxidative stress effects of the ischemia/reperfusion injury in young rat testis.

An optimized animal model for partial and total skin thickness burns studies

PURPOSE Development of an improved animal model for studying skin burns in rats. METHODS Twenty-four male Wistar rats were randomly assigned to four groups (n=6): G1-Control, G2- T100°C, G3-T150°C and G4-T200°C. Two 10 x 10 mm squares were outlined with a sterile surgical marker on each side and along the vertebral column using a prepared template positioned between the anterior and posterior limbs. G2-G4 rats were subjected to 100°C, 150°C and 200ºC thermal burns, respectively. G1 rats served as controls. Burns were inflicted by applying a copper plate connected to an electronic temperature controlling device to the dorsal skin of anesthetized rats. Four burns were produced on each animal (total area: 4 cm²/animal) leaving about 1 cm of undamaged skin between burn areas. Analgesia was administered during 24 h after burn injury by adding 30 mg codeine phosphate hemihydrate to 500 ml tap water. RESULTS The application of 100°C and 150ºC resulted in partial thickness skin burns with central reepithelialization of the burned area only at 100°C. In G4 group the whole thickness of the skin was injured without central reepithelialization. However, there was marginal reepithelialization in all groups. CONCLUSION The model studied is inexpensive and easily reproducible, enabling the achievement of controlled burns with partial or total impairment of the skin in experimental animals.

Enteral nutrition supplemented with l-glutamine in patients with systemic inflammatory response syndrome due to pulmonary infection

OBJECTIVE To evaluate the effect of enteral nutrition (EN) supplemented with l-glutamine on glycolytic parameters, inflammation, immune function, and oxidative stress in moderately ill intensive care patients with sepsis. METHODS Thirty patients received EN. Fifteen patients received EN supplemented with glutamine (30 g; GLN group) for 2 d followed by EN supplemented with calcium caseinate (30 g, CAS group), also over 2 d. The other 15 patients received EN with calcium caseinate (30 g; CAS group) for 2 d followed by EN with glutamine (30 g; GLN group), also over 2 days. One washout day with only EN was provided between every 2-d period of EN plus supplementation to all patients. Blood samples were taken before and after supplementation. RESULTS There were no changes in glycolytic parameters in either group. Leukocytes decreased in the two groups (from 13 650 to 11 500 in the CAS group, P = 0.019; from 12.850 to 11.000 in the GLN group, P = 0.046). Lymphocytes increased in the GLN group (from 954 to 1916, P < 0.0001) and were more numerous after glutamine supplementation (from 1916 to 1085, P < 0.0001, GLN versus CAS). No significant changes were observed in interleukin levels, but urea levels were higher in the GLN compared with the CAS group (50.0-47.0, P = 0.030). Glutathione plasma concentrations did not differ significantly between the groups. No significant changes were observed in the plasma glutamine and glutamate concentrations. CONCLUSIONS The EN supplemented with glutamine increased the lymphocyte count and helped to decrease lipid peroxidation but presented no effect on the antioxidant glutathione capacity and on cytokine concentrations or glycolytic parameters.

Dimethylsulfoxide attenuates ischemia-reperfusion injury in rat testis

PURPOSE To evaluate the protective role of dimethylsulfoxide (DMSO) in a rat model of testis ischemia/reperfusion (I/R). METHODS Twenty-four male Wistar rats were randomized in two equal groups. Control rats (G-1) received saline 2.0 ml intraperitoneally (ip) 21, 9 and 1 h before torsion. Experimental rats (G-2) received ip injections of 3% aqueous solution of DMSO, 0.1ml/10g body weight. Saline was added to complete 2.0ml when necessary. I/R injury was induced in anesthetized rats by torsion of the right testis lasting 3 hours. Testis and blood samples were collected at the end of ischemia (T-0) and 3 hours later (T-3) for assessment of testis malonaldehyde (MDA), reduced glutathione (GSH), and plasma total antioxidant power (TAP). RESULTS MDA levels decreased significantly in G-2 rats compared with G-1 animals in all time-points. GSH levels increased significantly in T-0 and T-3 time-points in DMSO pretreated rats compared with G-1 rats. GSH levels increased significantly during reperfusion in G-2 rats. TAP was similar in both groups denoting absence of systemic effects in this study. CONCLUSION Pretreatment with DMSO reduces testis lipid peroxidation and oxidative stress caused by torsion/detorsion of the testis.

O oleo essencial de Lippia gracilis Schauer, Verbenaceae, em ratos diabeticos

The essential oil from Lippia gracilis Schauer (Verbenaceae) leaves was examined by GC and GC-MS. Fifteen constituents were identified. Carvacrol, p-cymene and γ-terpinene were found to be the major components. In the in vitro study, 5% solution of the Lippia gracilis Schauer oil presented antibacterial activity against Staphylococcus aureus isolated from diabetic patients with infected ulcers. The study evaluated the antibacterial activity of the 5% solution of the Lippia gracilis Schauer oil on the experimental model of diabetic adult male albino Wistar rats with leaft pelvic limb infected by Staphylococcus aureus strain. In this experiment, 28 diabetic Wistar rats were used, randomly distributed in four different groups of seven rats, (G1-white; G2-negative control; G3-positive control and G4-test). When comparing group G4 with G3, it was observed that the 5% solution presented a reduced CFU/mL level showing the antibacterial effect of the oil 24 hours after the administration of the inoculum (S .aureus without Lippia gracilis Schauer 108 ±313 versus S.aureus with Lippia gracilis Schauer 13.28±4.03). The results were expressed as mean±S.E.M. One-way analysis of the variance (ANOVA) was used. The differences between the minimum inhibitory concentration in vitro test were determined by the Tukey test (p<0.05). The Newman-Keuls test with level of significance (p<0.05) was used to measure the results in vivo. The findings have shown that 5% solution of the Lippia gracilis Schauer oil presented antibacterial activity in vitro and in vivo.

Effects of L-alanyl-glutamine upon the blood and kidney biochemical parameters in the rat hind limb model of ischemia/reperfusion

PURPOSE To investigate the effects of l-alanyl-glutamine (Ala-Gln) intragastric administration upon blood and kidney metabolic parameters alterations in rats subjected to ischemia/reperfusion of hind limb. METHODS Forty-eight male rats were randomized in 2 groups offered via gavage either saline 2.0 mL (G-1) or Ala-Gln solution 0.75 mgKg-1(G-2) once a day at 7 AM during 7 days. One-hour after the last gavage (Day 7) all rats were submitted to ether anesthesia, laparotomy and clamping of the left iliac artery for 3 h. Kidney and blood samples were collected at the end of ischemic period (3 h) and at 1-3-6 h during reperfusion period for metabolites (pyruvate, lactate, glucose and ketone bodies) enzymatic analysis. ATP was also assayed in kidney samples. RESULTS Lactacemia and ketonemia were significantly increased in Ala-Gln treated rats during reperfusion. Kidney pyruvate concentrations were significantly decreased and tissue lactate concentrations were significantly increased during reperfusion (1 h and 3 h) in G-2 rats compared with respective controls. Glucose, ATP and ketone bodies concentrations were significantly increased in the kidney in L-Ala-Gln treated rats at 3 hours after reperfusion as compared to respective controls. CONCLUSIONS Unilateral hind limb ischemia in L-Ala-Gln pre-treated rats may induce increased lactacemia and increased kidney lactate concentrations, indicating increased glycolytic activity in renal medulla and in other peripheral tissues. Higher ketonemia during reperfusion may reflect a possible increase in ketogenesis due to lower insulin plasma concentration hepatic signaling as a result of increased glucose oxidation in peripheral tissues, caused by the intra-gastric administration of glutamine dipeptide, suggesting also decreased insulin resistance.

Preconditioning with L-alanyl-L-glutamine in a Mongolian Gerbil model of acute cerebral ischemia/reperfusion injury

PURPOSE To investigate the effect of L-alanyl-L-glutamine (L-Ala-Gln) preconditioning in an acute cerebral ischemia/reperfusion (I/R) model in gerbils. METHODS Thirty-six Mongolian gerbils (Meriones unguiculatus), (60-100g), were randomized in 2 groups (n=18) and preconditioned with saline 2.0 ml (Group-S) or 0.75g/Kg of L-Ala-Gln, (Group-G) administered into the femoral vein 30 minutes prior to I/R. Each group was divided into three subgroups (n=6). Anesthetized animals (urethane, 1.5g/Kg, i.p.) were submitted to bilateral occlusion of common carotid arteries during 15 minutes. Samples (brain tissue and arterial blood) were collected at the end of ischemia (T0) and after 30 (T30) and 60 minutes (T60) for glucose, lactate, myeloperoxidase (MPO), thiobarbituric acid reactive substances (TBARS), glutathione (GSH) assays and histopathological evaluation. RESULTS Glucose and lactate levels were not different in studied groups. However glycemia increased significantly in saline groups at the end of the reperfusion period. TBARS levels were significantly different, comparing treated (Group-G) and control group after 30 minutes of reperfusion (p<0.05) in cerebral tissue. Pretreatment with L-Ala-Gln promoted a significant increase in cerebral GSH contents in Group-G at T30 (p<0.001) time-point compared with Group-S. At T30 and T60, increased levels of GSH occurred in both time-points. There were no group differences regarding MPO levels. Pyknosis, presence of red neurons and intracellular edema were significantly smaller in Group-G. CONCLUSION Preconditioning with L-Ala-Gln in gerbils submitted to cerebral ischemia/reperfusion reduces oxidative stress and degeneration of the nucleus (pyknosis) and cell death (red neurons) in the cerebral tissue.

Electroacupuncture attenuates liver and kidney oxidative stress in anesthetized rats

PURPOSE Investigate the effects of a single electroacupuncture (EA) session at acupoints Zusanli (ST-36) and Zhongwan (CV-12) combined in regulating oxidative stress in liver and kidney in anesthetized rats. METHODS Eighteen healthy rats randomly assigned to 3 groups (n=6) were anesthetized intraperitoneally with ketamine (90 mg kg-1 body weight) + xylazine (10mg/kg body weight): G-1: Control (anesthesia), G-2: anesthesia+EA10 Hz and 10 mA, 10 Hz) applied to right ST-36 and CV-12 acupoints for 30 minutes. G-3 was likewise treated, using a tenfold higher frequency (100 Hz). G6PDH activity, malondialdehyde (MDA) and glutathione (GSH) levels were assayed spectrophotometrically. RESULTS Liver MDA and GSH concentrations increased significantly in rats submitted to EA 10Hz (p<0.01) and EA 100 Hz (p<0.001), compared with control G-1. Liver and kidney G6GPH activity decreased significantly in G-2 (p<0.01) and G-3 (p<0.001) compared with G-1 in EA100 Hz rats. A similar pattern was found in kidney G6PDH activity in EA10 Hz rats. CONCLUSION Single 30-minute EA 10/100 Hz session enhances lipid peroxidation and simultaneously reduces oxidative stress in liver and kidney tissues in a rat model.

Repercussões da L-alanil-glutamina sobre as concentrações de lactato e lactato desidrogenase (LDH) em pacientes com isquemia crítica dos membros inferiores submetidos a revascularização distal

PURPOSE: Investigate the repercussions of L-alanyl-glutamine in muscular tissue concentrations of lactate, and venous and arterial blood concentrations of LDH, in patients with critical ischemia of the lower limbs submitted to distal revascularization. METHODS: Sixteen adults (12 male/4 female) were distributed in 2 groups (1-Control/2-Experiment). Three hours after the intravenous injection of 250 ml of a 20% solution of L-alanyl-glutamine added to 750 ml of saline solution (Group 2); or 1000 ml of saline solution (Group 1), distal bypass was carried out under spinal anesthesia. Muscle and blood samples (arterial/venous) were collected at the beginning of the surgical procedure (TI), at the end (TF), and 10 and 20 minutes after re-establishment of blood flow. RESULTS: Significant reduction (p<0,05) of lactate concentration was observed in healthy muscle tissue in L-alanyl-glutamine treated patients in comparison to control group, at all times studied. There was a significant reduction (p <0,05) in venous concentrations of LDH in treated patients at all times studied (TI/TFV/T1V/T2V); and in arterial blood during reperfusion (T1A/T2A). CONCLUSIONS: 1. Decreased lactate concentrations in healthy skeletal muscle in patients treated with L-alanyl-glutamine suggests greater utilization of pyruvate for energy production than its conversion to lactate in Krebs cycle boosting aerobic glycolysis. 2. - Drop in venous blood concentrations of LDH in treated patients with L-alanyl-glutamine at all times during ischemia, and 10 and 20 minutes after reperfusion, also suggests augmented utilization of pyruvate for energy production via aerobic glycolysis.