CHARACTERISTICS OF ZINC-BINDING TO HUMAN RED-BLOOD-CELL MEMBRANES

The objective of the present study was to standardize the analysis of zinc binding on human red blood cell (RBC) membranes in 20 normal adults. The displacement studies revealed that at the maximal stable zinc concentration tested (600 muM), 57% (mean) of the bound Zn-65 was displaced and to displace half maximal Zn-65, the stable zinc concentration was 300 muM. Scatchard plots revealed two classes of binding sites for zinc on RBC membranes: one with higher affinity, Kd = 1.20 x 10(-5) M (site I), and the other with lower affinity, Kd = 2.77 x 10(-4) M (site II). Binding sites occupancy was 97% means and 58.5% means for sites I and 11, respectively. The displacement was affected by temperature, membrane protein concentration, freezing, thawing, and dialysis. Other metal cations, including Co++, Fe++, and Mn++, had very little effect on Zn-65 displacement, in contrast copper displaced Zn-65 from its binding sites on RBC membranes. Zinc binding to RBC membranes was rapid and readily reversible in a dynamic equilibrium with its binding sites. It is anticipated that this method will be applicable to studies of a wide variety of diseases specifically related to zinc metabolism in humans as well as in animals. (C) 1994 Wiley-Liss, Inc.

DAILY VARIATION IN PLASMA-CONCENTRATION OF FENCAMFAMINE AND STRIATAL DOPAMINE-RECEPTORS IN RATS

Fencamfamine (FCF) is a psychostimulant drug classified as an indirect dopamine agonist. In the present study we evaluated the daily variation in plasma FCF concentration and in striatal dopamine receptors. Adult male Wistar rats (250-300 g) maintained on a 12-h light/12-h dark cycle (lights on at 07:00 h) were used. Rats received FCF (10.0 mg/kg, ip) at 09:00, 15:00, 21:00 or 03:00 h and blood samples were collected 30 (N = 6) or 60 (N = 6) min after the injections. Plasma FCF was measured by gas chromatography using an electron capture detector. Two-way ANOVA showed significant differences in FCF concentration when blood samples were collected 30 min after the injection, and the highest value was obtained following injection 21:00 h. Moreover, at 15:00, 21:00 and 03:00h, plasma FCF levels were significantly lower 60 min after injection when compared to the 30-min interval. Two other groups of rats (N = 6) were decapitated at 09:00 or 21:00 h and the striata were dissected for the binding assays. The Bmax for [H-3]-spiroperidol binding to striatal membranes was higher at 21:00 h, without changes in affinity constant (Kd). In conclusion, plasma FCF levels and dopamine receptors undergo daily variation,a phenomenon that should be considered to explain the circadian time-dependent effects of FCF.

Effect of exercise mode on the blood lactate removal during recovery of high-intensity exercise

The aim of this study was to determine the effect of exercise mode on the blood lactate removal during recovery of high-intensity exercise. Nine male individuals performed the following tests in order to determine the blood lactate removal: Running - 2x200 m, the subjects ran at their maximum capacity, and rested 2 min between each bout. Swimming - 2x50 m, the subjects swam at their maximum capacity, and rested 2 min between each bout. Each test was realized on different days with three recovery modes: passive (sitting down), swimming, or running. Recovery exercise intensity was corresponding to the aerobic threshold. All recovery activities lasted 30 min. The two forms of active recovery were initiated 2 min after the end of high-intensity exercise and lasted 15 min, and were followed by 13 min of seated rest. After 1,7, 12,17, and 30 min of the end of high-intensity exercise, blood samples (25 mu l) were collected in order to determine the blood lactate concentration. By linear regression, between the logarithm of lactate concentration and its respective time of recovery, the half-time of blood lactate removal (t1/2) was determined. Time of high-intensity exercise and the lactate concentration obtained in the 1(st) min of recovery were not different between running and swimming. Passive recovery (PR) following running (R-PR=25.5+/-4.3 min) showed a t1/2 significantly higher than PR after swimming (S-PR=18.6+/-4.3 min). The t1/2 of the sequences running-running (R-R=13.0 min), running-swimming (R-S=12.9+/-3.8 min), swimming-swimming (S-S=13.2+/-2.8 min), and swimming-running (S-R=12.9+/-3.8 min) were significantly lower than the t1/2 of the R-PR and S-PR. There was no difference between the t1/2 of the sequences R-R R-S, and S-S. on the other hand the sequence S-R showed a t1/2 significantly lower than the sequences S-S and R-R. It was concluded that the two forms of active recovery determine an increase in the blood lactate removal, regardless of the mode of high-intensity exercise performed previously. Active recovery performed by the muscle groups that were not previously fatigued, can improve the blood lactate removal.

Characterization and quantification of blood cells from the umbilical cord of dogs

Background: Models for the study of hematopoietic stem cells in dogs provide important information for bone marrow transplantation in humans. Recent studies have reported the importance of human umbilical cord blood (UCB) as an alternative to allogenic bone marrow for hematopoietic reconstitution. However, there are no studies on the UCB cells of dogs. Objective: the aim of this experiment was to characterize and quantify the blood cells of the umbilical cord of dogs. Methods: the blood of the umbilical cord of 20 neonatal dogs, delivered at term, with a median gestation time of 58 days, was collected with a 5-mL syringe containing EDTA. Total RBC, WBC, and platelet counts, HCT, hemoglobin (Hgb) concentration, and RBC indices were determined using an automatic cell counter. The differential leukocyte count was determined manually in blood smears stained with May-Grunwald-Giemsa. Reticulocyte percentages were determined on blood smears stained with brilliant cresyl blue and counterstained with May-Grunwald Giemsa. Results: the MCHC and numbers of RBCs, WBCs, neutrophils, and eosinophils in UCB were lower as compared with reference values for the peripheral blood of healthy neonatal and adult dogs; whereas, the MCV and reticulocyte percentages were higher. Conclusion: Erythrocyte macrocytosis and hypochromasia in UCB were consistent with marked reticulocytosis and indicative of high erythropoietic activity. The results of this study are an important first step in the characterization of UCB from neonatal dogs.

The effects of ice-water storage on blood gas and acid-base measurements

Objective: To determine the effects of storage of arterial and venous blood samples in ice water on blood gas and acid-base measurements.Design: Prospective, in vitro, laboratory study.Setting: School of veterinary medicine.Subjects: Six healthy dogs.Measurements and main results: Baseline measurements of partial pressure of oxygen (PO2), partial pressure of carbon dioxide (PCO2), pH, hemoglobin concentration (tHb), oxyhemoglobin saturation, and oxygen content (ContO(2)) were made. Bicarbonate (HCO3) and standard base excess (SBE) were calculated. Arterial and venous blood samples were separated into 1 and 3 mL samples, anaerobically transferred into 3 mL plastic syringes, and stored in ice water for 6 hours. Measurements were repeated at 15, 30 minutes, and 1, 2, 4, and 6 hours after baseline measurements. Arterial (a) PO2 increased significantly from baseline after 30 minutes of storage in the 1 mL samples and after 2 hours in the 3 mL samples. Venous (v) PO2 was significantly increased from baseline after 4 hours in the 1 mL samples and after 6 hours in the 3 mL samples. The pHa significantly decreased after 2 hours of storage in the 1 mL samples and after 4 hours in the 3 mL samples. In both the 1 and 3 mL samples, pHv decreased significantly only after 6 hours. Neither the arterial nor the venous PCO2 values changed significantly in the 1 mL samples and increased only after 6 hours in the 3 mL samples. No significant changes in tHb, ContO(2), SBE, or HCO3 were detected.Conclusions: the PO2 of arterial and venous blood increased significantly when samples were stored in plastic syringes in ice water. These increases are attributable to the diffusion of oxygen from and through the plastic of the syringe into the blood, which occurred at a rate that exceeded metabolic consumption of oxygen by the nucleated cells.

Maximal lactate steady state concentration independent of pedal cadence in active individuals

The maximal lactate steady state (MLSS) is defined as the highest blood lactate concentration that can be maintained over time without a continual blood lactate accumulation. The objective of the present study was to analyze the effects of pedal cadence (50 vs. 100 rev min(-1)) on MLSS and the exercise workload at MLSS (MLSSworkload) during cycling. Nine recreationally active males (20.9 +/- 2.9 years, 73.9 +/- 6.5 kg, 1.79 +/- 0.09 m) performed an incremental maximal load test (50 and 100 rev min(-1)) to determine anaerobic threshold (AT) and peak workload (PW), and between two and four constant submaximal load tests (50 and 100 rev min(-1)) on a mechanically braked cycle ergometer to determine MLSSworkload and MLSS. MLSSworkload was defined as the highest workload at which blood lactate concentration did not increase by more than 1 mM between minutes 10 and 30 of the constant workload. The maximal lactate steady state intensity (MLSSintensity) was defined as the ratio between MLSSworkload and PW. MLSSworkload (186.1 +/- 21.2 W vs. 148.2 +/- 15.5 W) and MLSSintensity (70.5 +/- 5.7% vs. 61.4 +/- 5.1%) were significantly higher during cycling at 50 rev min(-1) than at 100 rev min(-1), respectively. However, there was no significant difference in MLSS between 50 rev min(-1) (4.8 +/- 1.6 mM) and 100 rev min(-1) (4.7 +/- 0.8 mM). We conclude that MLSSworkload and MLSSintensity are dependent on pedal cadence (50 vs. 100 rev min(-1)) in recreationally active individuals. However, this study showed that MLSS is not influenced by the different pedal cadences analyzed.

LH and FSH concentration and follicular development in Nellore heifers submitted to fixed-time artificial insemination protocols with different progesterone concentrations

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Influence of exercise mode and maximal lactate-steady-state concentration on the validity of OBLA to predict maximal lactate-steady-state in active individuals

The aim of this study was to analyze the effects of exercise mode on the validity of onset of blood lactate accumulation (OBLA-3.5-mM fixed blood lactate concentration) to predict the work-rate at maximal lactate steady state (MLSSwork-rate). Eleven recreationally active mates (21.3 +/- 2.9 years, 72.8 +/- 6.7 kg, 1.78 +/- 0.1 m) performed randomly incremental tests to determine OBLA (stage duration of 3 min), and 2 to 4 constants work-rate exercise tests to directly determine maximal lactate steady state parameters on a cycle-ergometer and treadmill. For both exercise modes, the OBLA was significantly correlated to MLSSwork-rate, (cycling: r = 0.81 p = 0.002; running: r = 0.94, p < 0.001). OBLA (156.2 +/- 41.3 W) was lower than MLSSwork-rate (179.6 +/- 26.4 W) during cycling exercise (p = 0.007). However, for running exercise, there was no difference between OBLA (3.2 +/- 0.6 m s(-1)) and MLSSwork-rate (3.1 +/- 0.4 m s(-1)). The difference between OBLA and MLSSworkrate on the cycle-ergometer (r = 0.86; p < 0.001) and treadmill (r = 0.64; p = 0.048) was significantly related to the specific MLSS. We can conclude that the validity of OBLA on predicting MLSSwork-rate is dependent on exercise mode and that its disagreement is related to individual variations in MLSS. (C) 2007 Sports Medicine Australia. Published by Elsevier Ltd. All rights reserved.

The workload and plasma ion concentration in a training match session of high-goal (elite) polo ponies

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

HPLC determination of flumethrin, deltamethrin, cypermethrin, and cyhalothrin residues in the milk and blood of lactating dairy cows

A procedure to determine residue concentrations of synthetic pyrethroid insecticides (flumethrin, deltamethrin, cypermethrin and cyhalothrin) in the milk and blood of lactating dairy cows was developed. Extraction was performed with acetonitrile, n-hexane partitioning, and silica gel column cleanup with n-hexane and diethyl ether. Analysis was carried out by high- performance liquid chromatography and ultraviolet detection. Recovery of the four pyrethroids averaged 78 to 91% with a minimum detectable concentration of 0.001 mg/kg. The method was reproducible and sensitive.

Evaluation of a protein deficient diet in rats through blood oxidative stress biomarkers

Protein malnutrition leads to functional impairment in several organs, which is not fully restored with nutritional recovery. Little is known about the role of oxidative stress in the genesis of these alterations. This study was designed to assess the sensitivity of blood oxidative stress biomarkers to a dietary protein restriction. Male Wistar rats were divided into two groups, according to the diet fed from weaning (21 days) to 60 day old: normal protein (17% protein) and low protein (6% protein). Serum protein, albumin, free fatty acid and liver glycogen and lipids were evaluated to assess the nutritional status. Blood glutathione reductase (GR) and catalase (CAT) activities, plasma total sulfhydryl groups concentration (TSG) as well as plasma thiobarbituric acid reactive substances (TBARs) and reactive carbonyl derivatives (RCD) were measured as biomarkers of the antioxidant system and oxidative damage, respectively. The glucose metabolism in soleus muscle was also evaluated as an index of stress severity imposed to muscular mass by protein malnutrition. No difference was observed in muscle glucose metabolism or plasma RCD concentration between both groups. However, our results showed that the low protein group had higher plasma TBARs (62%) concentration and lower TSG (44%) concentration than control group, indicating increased reactive oxygen species production in low protein group. The enhancement of erythrocyte GR (29%) and CAT (28%) activities in this group also suggest an adaptation to the stress generated by the protein deficiency. Taken together, the results presented here show that the biomarkers used were able to reflect the oxidative stress level induced by this specific protein deficient diet.

Effect of remifentanil hydrochloride administered via constant rate infusion on the minimum alveolar concentration of isoflurane in cats

Objective - To evaluate the effects of increasing doses of remifentanil hydrochloride administered via constant rate infusion (CRI) on the minimum alveolar concentration (MAC) of isoflurane in cats. Animals - 6 healthy adult cats. Procedures - For each cat, 2 experiments were performed (2-week interval). On each study day, anesthesia was induced and maintained with isoflurane; a catheter was placed in a cephalic vein for the administration of lactated Ringer's solution or remifentanil CRIs, and a catheter was placed in the jugular vein for collection of blood samples for blood gas analyses. On the first study day, individual basal MAC (MAC Basal) was determined for each cat. On the second study day, 3 remifentanil CRIs (0.25, 0.5, and 1.0 μg/kg/min) were administered (in ascending order); for each infusion, at least 30 minutes elapsed before determination of MAC (designated as MAC R0.25, MAC R0.5, and MAC R1.0, respectively). A 15-minute washout period was allowed between CRIs. A control MAC (MAC Control) was determined after the last remifentanil infusion. Results - Mean ± SD MAC Basal and MAC Control values at sea level did not differ significantly (1.66 ± 0.08% and 1.52 ± 0.21%, respectively). The MAC values determined for each remifentanil CRI did not differ significantly. However, MAC R0.25, MAC R0.5, and MAC R1.0, were significantly decreased, compared with MAC Basal, by 23.4 ± 79%, 29.8 ± 8.3%, and 26.0 ± 9.4%, respectively. Conclusions and Clinical Relevance - The 3 doses of remifentanil administered via CRI resulted in a similar degree of isoflurane MAC reduction in adult cats, indicating that a ceiling effect was achieved following administration of the lowest dose.

Experimental infection of commercial layers using a Salmonella enterica sorovar Gallinarum strain: Blood serum components and histopathological changes

The purpose of the study was to evaluate the blood serum components and histopathological findings of commercial layers experimentally infected with Salmonella Gallinarum (SG), the microorganism responsible for the fowl typhoid. 180 commercial layers were distributed into three groups (G): G1 and G2 received 0.2mL of inoculum containing 3.3x10 8 and 3.3x10 5 CFU of resistant SG to the nalidix acid (Nal r)/mL, respectively, directly into their crops; G3 did not receive the inoculum (control group). The birds were inoculated when they were 5 days old and the euthanasia was performed 24 hours before and after infection and 3, 5, 7 and 10 days after the administration of the inoculum. In each day of collection, blood samples were obtained for biochemical tests of the blood serum besides macroscopic and histopathological examination of the birds. Data were submitted to analysis of variance by the SAS statistical program and the means were compared by Tukeýs test (P<0,05). In the serum biochemical profile it was observed that the infection interfered in the values of total protein, albumin, calcium, phosphorus, cholesterol, triglycerides, GGT and ALT in the infected groups. The macroscopic examination showed hepatomegaly, alteration of the hepatic color and hemorrhagic spots in the kidneys of animals from G1. The histopathology showed degeneration of hepatocytes in G1 and G2 although other lesions like multifocal hepatic necrosis and inflammatory infiltrate on the liver and kidneys were restricted to G1. The alterations were more evident on G1 which received a higher concentration of bacteria/mL when compared to G2. The results showed that the correlation between biochemical alterations and macroscopic and histopathological lesions can assist the comprehension of the pathophysiology of fowl typhoid, supplying important information for the diagnosis and prognosis of this disease.

Simple method for culture of peripheral blood lymphocytes of Testudinidae.

We developed and optimized a simple, efficient and inexpensive method for in vitro culture of peripheral blood lymphocytes from the Brazilian tortoise Chelonoidis carbonaria (Testudinidae), testing various parameters, including culture medium, mitogen concentration, mitotic index, culture volume, incubation time, and mitotic arrest. Peripheral blood samples were obtained from the costal vein of four couples. The conditions that gave a good mitotic index were lymphocytes cultured at 37°C in minimum essential medium (7.5 mL), with phytohemagglutinin as a mitogen (0.375 mL), plus streptomycin/penicillin (0.1 mL), and an incubation period of 72 h. Mitotic arrest was induced by 2-h exposure to colchicine (0.1 mL), 70 h after establishing the culture. After mitotic arrest, the cells were hypotonized with 0.075 M KCl for 2 h and fixed with methanol/acetic acid (3:1). The non-banded mitotic chromosomes were visualized by Giemsa staining. The diploid chromosome number of C. carbonaria was found to be 52 in females and males, and sex chromosomes were not observed. We were able to culture peripheral blood lymphocytes of a Brazilian tortoise in vitro, for the preparation of mitotic chromosomes.

Effect of dexmedetomidine on the minimum alveolar concentration of isoflurane in cats

This study reports the effects of dexmedetomidine on the minimum alveolar concentration of isoflurane (MAC iso) in cats. Six healthy adult female cats were used. MAC iso and dexmedetomidine pharmacokinetics had previously been determined in each individual. Cats were anesthetized with isoflurane in oxygen. Dexmedetomidine was administered intravenously using target-controlled infusions to maintain plasma concentrations of 0.16, 0.31, 0.63, 1.25, 2.5, 5, 10, and 20ng/mL. MAC iso was determined in triplicate at each target plasma dexmedetomidine concentration. Blood samples were collected and analyzed for dexmedetomidine concentration. The following model was fitted to the concentration-effect data: where MAC iso.c is MAC iso at plasma dexmedetomidine concentration C, MAC iso.0 is MAC iso in the absence of dexmedetomidine, I max is the maximum possible reduction in MAC iso, and IC 50 is the plasma dexmedetomidine concentration producing 50% of I max. Mean±SE MAC iso.0, determined in a previous study conducted under conditions identical to those in this study, was 2.07±0.04. Weighted mean±SE I max, and IC 50 estimated by the model were 1.76±0.07%, and 1.05±0.08ng/mL, respectively. Dexmedetomidine decreased MAC iso in a concentration-dependent manner. The lowest MAC iso predicted by the model was 0.38±0.08%, illustrating that dexmedetomidine alone is not expected to result in immobility in response to noxious stimulation in cats at any plasma concentration. © 2011 Blackwell Publishing Ltd.