Paulo Roberto Leitão de Vasconcelos

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.

The safety of oral use of L-glutamine in middle-aged and elderly individuals.

OBJECTIVE To evaluate the safety of nutraceutical oral administration of L-glutamine (L-Gln) in middle-aged and elderly individuals. METHODS In this randomized, crossover, double-blind clinical study, 30 residents of a long-term-care institution, selected according to a modified SENIEUR protocol (Working Party of the EURAGE Concerted Action Programme on Ageing of the European Community), were studied. Fourteen subjects received orally 0.5 g kg(-1) d(-1) of L-Gln and 16 received calcium caseinate for 14 d, followed by a 5-d washout. Supplements were switched for the second 14-d trial. Laboratory tests for hepatic and renal functions and ammonemia were performed and the estimated glomerular filtration rate (eGFR) was calculated. RESULTS Of the 30 subjects, 16 were men, mean age was 69+/-8.8 y, average weight was 61.8+/-14.2 kg, and mean serum albumin was 4.0+/-0.3g/dL. Neither adverse clinical effects nor clinically significant laboratory changes were noted during L-Gln supplementation. There was no difference in ammonemia between the groups. There were statistically but not clinically significant increases in plasma urea nitrogen and creatinine concentrations. There was no significant decrease in eGFR during calcium caseinate supplementation (-2.9%). The eGFR decreased significantly after L-Gln supplementation (-13.3%) but well below the 25% limit for biologic significance. CONCLUSION Increases in serum urea nitrogen and creatinine and decrease in eGFR are probably due to difficulties by older kidneys in metabolizing the supplemented protein sources. Although not clinically significant, those alterations impose a rigorous control on the evaluation parameters of renal function during oral L-Gln supplementation, with doses of 0.5 g kg(-1) d(-1) in middle-aged and elderly individuals.

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.

Extrato de Passiflora edulis na cicatrização de anastomose colônica em ratos: estudo morfológico e tensiométrico

INTRODUCTION: Investigation of new substances with therapeutic effects have been done trying to isolate, extract or purify new compounds of vegetable origin. The Passiflora edulis (maracuja) species from the Plassifloracia family, originated from the tropical and subtropical regions of the american continent, is found all over Brazil. It is commonly used as a sedative, painkiller and anti-inflammatory drug and also for the treatment of skin wounds, lesions and Erisipelae. PURPOSE: To evaluate the wound healing in colonic anastomosis in rats that received an hydro-alcoholic extract of Passiflora edulis peri-operatively. METHOD: 40 wistar rats were used distributed into two groups of 20 rats each, named: Passiflora edulis group (GP) and control group (GC). The rats of each group were separated into two subgroups of 10 animals each and were evaluated on the 3rd and 7th postoperative days. The surgical procedure consisted of a section of the left colon, 5 cm above the peritoneal reflexion with preservation of the vascular elements. Intestinal continuity was restored by an end-to-end single layer anastomosis. The Passiflora edulis group received an intraperitoneal application of the hydro-alcoholic extract in the dosage of 250 mg/kg. The control-group received one intraperitoneal dose of a saline with the same volume of the GP. The parameters evaluated were: macroscopic aspects of the wall and abdominal cavity, perianastomotic (adherences), bursting pressure, inflammatory tissue reaction on the anastomotic wound. RESULTS: The macroscopic aspects did not differ between the groups. No rupture in the anastomotic wound was seen in any rat. Regarding the bursting pressure, it was noticed that the average pressure was significantly higher in the subgroup that received the Passiflora edulis extract on the 3rd day (P3) (42,6 ± 17,8 mmHg vs. 25,4 ± 14,1 mmHg, p=0,028), as compared to the control sub-group (C3). However, on the 7th day, bursting pressure was similar in both groups (p=0.447). Rats from the C7 sub-group had a mean bursting pressure of 203,0 ± 50,0 mmHg vs. 187,3 ± 39,5 mmHg in the C7 sub-group. In the histologic analysis the polimorphic nuclear cells were more frequent in the C3 group, with significant differences (p=0,034). The monomorphic nuclear cells (MMN) and the fibrobastic proliferation were more frequent in the P3 sub-group with a significant difference, p=0,02 to MMN, and p=0,001 to the fibroblastic proliferation. On the 7th day there was a significant difference in all histologic criteria stained by hematoxin-eosin and Masson Trichomic (p<0,05) in the sub-group that received the Passiflora edulis extracts. CONCLUSION: The peri-operative administration of the hydro-alcoholic extract of Passiflora edulis has a positive influence on the healing of colonic anastomosis in rats.

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.

Modelo de tumor experimental em rim de ratos

Walker carcinossarcoma 256 has a great interest as experimental model for studies on tumoral biology. OBJECTIVE: develop a kidney tumor model to be used in the evaluation of the biological behavior of neoplasms in vitro and in vivo environments. METHODS: twenty adult male Wistar rats weighting between 250-300 g were obtained from the Federal University of the Ceara Experimental Surgery Laboratory. Upon ether anesthesia, the right kidney of each animal was accessed through a supraumbelical incision and inoculated with a solution containing 3 x 105 tumor cells (Walker 256 carcinossarcoma tumor cells). Following anesthetic recovery the rats were returned to their cages, housed individually and allowed to eat and drink ad libitum. RESULTS: the post-operative average survival was 14 days. Tumor growth was encountered in all animals. The rats exhibited destruction of the kidney, invasion of the adjacent structures, but absence of metastasis. CONCLUSION: the kidney Walker tumor model proved to be an excellent tool for experimental studies on tumor behavior, due to its rapid growth and its easy reproduction.