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The in vitro effect of different concentrations of beta-hydroxybutyric acid (betaHBA) on bovine milk leukocytes was examined. betaHBA level similar to those found in cows with clinical ketosis induced a significant inhibitory effect on the nitroblue tetrazolium reduction as a mean of assaying the metabolic integrity of macrophages after the phorbol-mirystate- acetate and opsonized zymosan stimulation. In the same way, the H2O2 production after stimulation with both soluble and particulate agents decreased significantly in 33 and 26%, respectively, compared with cells incubated without ketone bodies. This result suggests a possible fault in the microbicidal oxidative activity. The macrophage phagocytosis also decreased in cells treated with different betaHBA concentrations, in relation to that obtained from control cells. Neutrophils migration in agarose was determined, and the mean chemotactic response was higher when the cells were incubated with lower level or absence of ketone bodies. Considering the determined differences, we hypothesize that abnormally high levels of ketone bodies could produce a direct effect on leukocyte membranes. The induction of some modification on the receptor structure impairment the interaction ligand-receptor and this may be, in part, responsible for the higher susceptibility to local infections in mammary gland during subclinical and clinical ketosis.

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The role of leptin as an energetic state indicator in cattle was evaluated correlating different nutritional conditions and serum leptin levels. Two experiments were developed. Experiment I: Polled Hereford heifers were fed in optimum conditions and assigned randomly to one of three treatment diets: grazing, grazing plus supplementation and feedlot. Experiment II: cattle were subjected to feed restriction (R). Serum leptin levels were measured at pre and post-feeding moments. Using serum free fatty acid (FFA), beta-OH Butyrate (beta-OHB) and protein concentration as serum energetic indicators in both experiments. In experiment I leptin values were significantly different between pre and post-feeding moments, similar differences were found among groups when the pre-feeding and post-feeding values were compared. These differences may be considered as changes associated to the control of food intake. In experiment II, the serum leptin levels in Group R were significantly lower. The poor energetic status of animals from group R was demonstrated by the serum FFA, beta-OHB and protein concentrations. These results suggest that the energetic status of animals is correlated with the serum leptin levels, tending to maintain the energetic homeostasis.

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The activity of several cuproenzymes in relation to the immune system was examined in serum and blood cells from bovines with molybdenum-induced copper deficiency. Five female cattle were given molybdenum (30 ppm) and sulfate (225 ppm) to induce experimental secondary copper deficiency. Ceruloplasmin activity was determined in serum. The Cu,Zn-superoxide dismutase and cytochrome c oxidase activities were measured in peripheral blood lymphocytes, neutrophils, and monocyte-derived macrophages. Copper deficiency was confirmed from decreased serum copper levels and the animals with values less than 5.6 micromol/L were considered deficient. The content of intracellular copper decreased between 40% and 70% in deficient cells compared with the controls. In copper-deficient animals, the serum ceruloplasmin activity decreased to half of the control value. Both of them, the Cu,Zn-superoxide dismutase and the cytochrome c oxidase activities, undergo a significant reduction in leukocytes, showing differences among diverse cell populations. We concluded that the copper deficiency alters the activity of several enzymes, which mediate antioxidant defenses and ATP formation. These effects may impair the cell immune functionality, affecting the bactericidal capacity and making the animals more susceptible to infection.

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The effect of molybdenum-induced copper deficiency on monocyte-derived macrophage function was examined. Five female calves were given molybdenum (30 ppm) and sulphate (225 ppm) to induce experimental secondary copper deficiency. Oxidant production by bovine macrophages was measured after stimulation with phorbol myristate acetate (PMA) and opsonized zymosan (OpZ). Lipoperoxidative effects inside of macrophage, superoxide dismutase activity, superoxide anion and hydrogen peroxide formation were determined. Copper deficiency was confirmed from decreased serum copper levels, and animals with values less than 5.9 micromol/l were considered deficient. The content of intracellular copper decreased about 40% in deficient cells compared with the controls. The respiratory burst activity determined by nitroblue tetrazolium reduction was significantly impaired with both stimulants used. Superoxide anion formation was less affected than hydrogen peroxide generation. In addition, increased lipid peroxidation was observed. It could be concluded that the effect of these changes may impair the monocyte-derived macrophage function in the immune system.

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1. The enantioselective liver microsomal sulphoxidation of the benzimidazole anthelmintic, albendazole (ABZ), by cattle liver microsomes has been investigated. The influence of nutritional condition on this biotransformation process was also characterized. 2. ABZ was oxidized to its sulphoxide metabolite (ABZSO) in a NADPH concentration-dependent reaction and the (+) and (-) ABZSO enantiomers formed were identified. 3. Vmax (0.27 nmol ABZSO formed per min x mg(-1) microsomal protein) and Km (15.10 microM) for ABZ sulphoxidation by cattle liver microsomes were obtained. Different Vmax (0.11 and 0.16 nmol x min(-1) x mg(-1)) and Km (9.40 and 26.70 microM) characterized the enantioselective formation of (+) and (-) ABZSO antipodes, respectively. 4. Free fatty acid (FFA) concentrations and beta-hydroxybutyrate concentrations (beta-OHB) in serum and liver homogenates were significantly higher in feed-restricted (poor nutritional condition) compared with control animals in an optimal nutritional status. Serum protein concentrations and liver cytosolic glucose 6-phosphate dehydrogenase (G6PD) activity were significantly lower in the feed-restricted compared with control calf. 5. Animal nutritional condition affected the pattern of ABZ sulphoxidation. A higher Km for (total) ABZSO and (+) ABZSO production was observed in the calf subjected to a period of undernutrition. 6. A nutritionally induced impairment in the affinity of microsomal mixed-function oxidases responsible of ABZ oxidation may be responsible for the observed changes in the liver microsomal sulphoxidation of ABZ in the feed-restricted calf. Furthermore, undernutrition may affect primarily the FMO-mediated formation of (+) ABZSO. These in vitro observations agree with the changes observed in vivo following the administration of ABZ to the calf subjected to a dietary restriction.

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The objectives of this study were to evaluate the activity of antioxidant systems in the hepatic tissues of steers experimentally subjected to a restricted diet. Hence, the activities of superoxide dismutase (SOD) and reduced glutathione (GSH) and lipid peroxide levels were measured. Nine male Holstein steers were used. They were separated in two groups: three steers in group 1 (control) and six steers in group 2, which were subjected to a restricted diet that covered only 60% of the maintenance requirements. After 30 days, the animals in both groups were killed and studies were completed. Both the CuZn-SOD and the glucose-6-phosphate dehydrogenase activities were significantly decreased (p < 0.05) in group 2, reaching 68% and 60%, respectively, of the concentrations found in group 1. The concentration of GSH in group 2 was 6.71+/-0.9 nmol/mg protein, which was significantly lower (p < 0.01) than that of the controls, 25.7+/-2.4 nmol/mg protein. In addition, the lipid peroxide levels were significantly increased (p < 0.01) in group 2, being 50-60% higher than that in group 1. These results showed that the poor nutritional status caused modifications to the enzymatic antioxidant systems, with a lower ability to reduce oxidative compounds and a state of lipid peroxidation.

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Six female cattle were given molybdenum (30 ppm) and sulphate (225 ppm) to induce experimental secondary copper deficiency. The total and differential leukocyte numbers and lymphocyte subpopulations were counted and the neutrophil activity was assessed by means of nitroblue tetrazolium reduction and phagocytosis of sheep erythrocytes. The serum caeruloplasmin activity and concentration were also determined. Copper deficiency was confirmed from decreased serum copper levels, the animals with values less than 5.9 mumol/L being considered deficient. Total leukocyte numbers were not affected by the copper deficiency. However, differential counts showed a marked increase in monocyte subpopulations, a significant decrease in B lymphocytes and reduced neutrophil activity. The serum caeruloplasmin activity was decreased about 50%, but the total serum protein concentration was less altered. We concluded that the effect of these changes on the animals' immune competence may contribute to a greater incidence of infectious diseases in copper-deficient cattle.

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To explain the cytotoxicity of excessive free radical production in the liver of rats, the lipoperoxidation in subcellular structures and some antioxidants systems were evaluated. We measured Cu-Zn superoxide dismutase (Cu-Zn-SOD) activity, reduced glutathione (GSH) levels and lipid peroxidation in homogenates and subcellular fractions of hepatocytes. Female Wistar rats were given a 0,2 per cent solution of CUSO4 in water, to induce the Cu toxicity. Serum copper levels and acid phosphatase (AP) activity were determined at frequent intervals. Six treated rats were euthanased to the twelfth week of begun the assay. During the sixteenth week, at time of the increase of serum AP activity the others treated rats also were killed. We found high liver Cu content and evidence of lipid peroxidation. In whole homogenate, mitochondrial and microsomal fractions, the thiobarbituric acid reacting substances were increased. This was correlated with an increase in the Cu-Zn-SOD activity and with decrease of the GSH levels. It could be argued that high copper status might have increased the Cu-Zn-SOD activity and induced lowest levels of GSH. Additionally, lipid peroxidation was induced by Cu-overload.

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The present study was undertaken to investigate the capacity to generate reducing equivalents in erythrocytes from experimentally copper-poisoned sheep. Ten ewes were dosed orally with CuSO4 to induce the Cu toxicity. Copper dosing was stopped at the first day of hemolysis. The activity of glucose-6-phosphate dehydrogenase (G6PD) in the erythrocytes, the levels of reduced glutathione (GSH) and glucose (in serum and erythrocytes) was examined at frequent intervals. The copper-poisoned sheep had reduced levels (25-35% less) of glucose in serum and erythrocytes than controls. The activity of G6PD in erythrocytes from sheep was 50-60% of typical levels found in human erythrocytes. Immediately before the hemolytic period, the copper-poisoned sheep showed decreased activity of G6PD, declining to 65% of the initial activity. In addition, we found decreased blood levels of reduced GSH in copper-poisoned sheep. There appears to be a relationship between decreased capacity to generate reducing equivalents and the overload of copper in sheep erythrocytes.

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Using clinical, functional and biochemical criteria, we studied the red blood cell membrane of nine caucasic patients from four families with hereditary elliptocytosis (HE). From a clinical point of view, seven cases were classified as compensated mild HE in whom anemia and splenomegaly are absent and reticulocytosis is slightly elevated or normal. Two cases were uncompensated mild HE with anemia, reticulocytosis, splenomegaly and erythrocytic fragmentation. (Table 1). Three individuals from one family displayed a significant reduction of protein 4.1 by polyacrylamide gel electrophoresis; one of them was uncompensated HE. The patterns of limited tryptic digestion and dimer/tetramer proportion of spectrin were normal. The study of red cell deformability by ectacytometry revealed that the cell deformability under isotonic conditions was decreased in all HE patients and the curve obtained had trapezoidal shape (Fig. 3). We found that the deformability index correlated well with the degree of anemia, but no correlation was observed between clinical findings, morphological phenotype and specific molecular etiology. According to our knowledge, this is the first report on molecular and functional studies in HE in Argentina.

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Using clinical, functional and biochemical criteria, we studied the red blood cell membrane of nine caucasic patients from four families with hereditary elliptocytosis (HE). From a clinical point of view, seven cases were classified as compensated mild HE in whom anemia and splenomegaly are absent and reticulocytosis is slightly elevated or normal. Two cases were uncompensated mild HE with anemia, reticulocytosis, splenomegaly and erythrocytic fragmentation. (Table 1). Three individuals from one family displayed a significant reduction of protein 4.1 by polyacrylamide gel electrophoresis; one of them was uncompensated HE. The patterns of limited tryptic digestion and dimer/tetramer proportion of spectrin were normal. The study of red cell deformability by ectacytometry revealed that the cell deformability under isotonic conditions was decreased in all HE patients and the curve obtained had trapezoidal shape (Fig. 3). We found that the deformability index correlated well with the degree of anemia, but no correlation was observed between clinical findings, morphological phenotype and specific molecular etiology. According to our knowledge, this is the first report on molecular and functional studies in HE in Argentina.

RESUMO

Using clinical, functional and biochemical criteria, we studied the red blood cell membrane of nine caucasic patients from four families with hereditary elliptocytosis (HE). From a clinical point of view, seven cases were classified as compensated mild HE in whom anemia and splenomegaly are absent and reticulocytosis is slightly elevated or normal. Two cases were uncompensated mild HE with anemia, reticulocytosis, splenomegaly and erythrocytic fragmentation. (Table 1). Three individuals from one family displayed a significant reduction of protein 4.1 by polyacrylamide gel electrophoresis; one of them was uncompensated HE. The patterns of limited tryptic digestion and dimer/tetramer proportion of spectrin were normal. The study of red cell deformability by ectacytometry revealed that the cell deformability under isotonic conditions was decreased in all HE patients and the curve obtained had trapezoidal shape (Fig. 3). We found that the deformability index correlated well with the degree of anemia, but no correlation was observed between clinical findings, morphological phenotype and specific molecular etiology. According to our knowledge, this is the first report on molecular and functional studies in HE in Argentina.

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Se estudiaron nueve pacientes (cuatro familias) con eliptocitosis herediataria (EH). Desde el punto de vista clínico, se identificaron dos tipos de presentación: siete pacientes fueron formas compensadas y dos (de 7 y 63 años) pertenecientes a distintas familias mostraron anemia, esplenomegalia, reticulocitosis y fragmentación globular. En una familia se comprobó una disminución del stock total de la proteína 4.1 en la electroforesis de proteína de membrana en gel de poliacrilamida con SDS (SDS-PAGE). Uno de sus miembros se comprobó como forma descompensada. El estudio de dímeros y tetrámeros y la digestión tríptica de espectrina fue normal en todos los casos. En la determinación de la deformabilidad en función de la osmoralidad con el ectacitómetro se halló una buena correlación entre el índice de deformabilidad (ID) y el grado de anemia. De acuerdo a los resultados de este estudio pudimos confirmar la gran heterogeneidad clínica de la EH aún en miembros de una misma familia (AU)

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Se estudiaron nueve pacientes (cuatro familias) con eliptocitosis herediataria (EH). Desde el punto de vista clínico, se identificaron dos tipos de presentación: siete pacientes fueron formas compensadas y dos (de 7 y 63 años) pertenecientes a distintas familias mostraron anemia, esplenomegalia, reticulocitosis y fragmentación globular. En una familia se comprobó una disminución del stock total de la proteína 4.1 en la electroforesis de proteína de membrana en gel de poliacrilamida con SDS (SDS-PAGE). Uno de sus miembros se comprobó como forma descompensada. El estudio de dímeros y tetrámeros y la digestión tríptica de espectrina fue normal en todos los casos. En la determinación de la deformabilidad en función de la osmoralidad con el ectacitómetro se halló una buena correlación entre el índice de deformabilidad (ID) y el grado de anemia. De acuerdo a los resultados de este estudio pudimos confirmar la gran heterogeneidad clínica de la EH aún en miembros de una misma familia

Assuntos