Sulfur-induced polioencephalomalacia (PEM) is an important disease affecting cattle in certain geographical regions. However, the pathogenesis of brain damage is not completely understood. We previously observed that excess dietary sulfur may influence thiamine status and altered thiamine metabolism may be involved in the pathogenesis of sulfur-induced PEM in cattle. In this study, we evaluated the activities of thiamine-dependent enzymes [α-ketogluterate dehydrogenase (α-KGDH) and pyruvate dehydrogenase (PDH)] and cytochrome c oxidase (COX) in the cerebral cortex of sulfur-induced PEM-affected cattle (n = 9) and clinically normal cattle (n = 8, each group) exposed to low or high dietary sulfur [LS = 0.30% versus HS = 0.67% sulfur on a dry matter (DM) basis]. Enzyme activities in PEM brains were measured from the brain tissue regions and examined using ultraviolent (UV) light illumination to show fluorescence or non-fluorescence regions. No gross changes under regular or UV light, or histopathological changes indicative of PEM were detected in the brains of cattle exposed to LS or HS diets. The PDH, α-KGDH, and COX activities did not differ between LS and HS brains, but all enzymes showed significantly lower (P < 0.05) activities in UV-positive region of PEM brains compared with LS and HS brains. The UV-negative regions of PEM brain had similar PDH activities to LS and HS brains, but the activities of α-KGDH and COX were significantly lower than in LS and HS brains. The results of this study suggest that reduced enzyme activities of brain PHD, α-KGDH, and COX are associated with the pathogenesis of sulfur-induced PEM.

OBJECTIVE To assess the discriminatory value for corticosteroid-induced alkaline phosphatase (CiALP) activity and other variables that can be measured routinely on a CBC and biochemical analysis for the diagnosis of hypoadrenocorticism in dogs. SAMPLE Medical records of 57 dogs with confirmed hypoadrenocorticism and 57 control dogs in which hypoadrenocorticism was suspected but ruled out. PROCEDURES A retrospective case-control study was conducted. Dogs were included if a CBC and complete biochemical analysis had been performed. Dogs with iatrogenic hypoadrenocorticism and dogs treated previously with glucocorticoids were excluded. Cortisol concentration for dogs with hypoadrenocorticism was ≤ 2 μg/dL both before and after ACTH administration. Cortisol concentration for control dogs was > 4 μg/dL before or after ACTH administration. RESULTS Area under the receiver operating characteristic (ROC) curve for CiALP activity was low (0.646; 95% confidence interval, 0.494 to 0.798). Area under the ROC curve for a model that combined the CiALP activity, Na-to-K ratio, eosinophil count, activity of creatine kinase, and concentrations of SUN and albumin was high (0.994; 95% confidence interval, 0.982 to 1.000). Results for this model could be used to correctly classify all dogs, except for 1 dog with hypoadrenocorticism and no electrolyte abnormalities. CONCLUSIONS AND CLINICAL RELEVANCE CiALP activity alone cannot be used as a reliable diagnostic test for hypoadrenocorticism in dogs. Combined results for CiALP activity, Na-to-K ratio, eosinophil count, creatine kinase activity, and concentrations of SUN and albumin provided an excellent means to discriminate between hypoadrenocorticism and diseases that mimic hypoadrenocorticism.

Objective—To determine the effect of large colon ischemia and reperfusion on concentrations of the inflammatory neutrophilic protein calprotectin and other clinicopathologic variables in jugular and colonic venous blood in horses. Animals—6 healthy horses. Procedures—Horses were anesthetized, and ischemia was induced for 1 hour followed by 4 hours of reperfusion in a segment of the pelvic flexure of the large colon. Blood samples were obtained before anesthesia, before induction of ischemia, 1 hour after the start of ischemia, and 1, 2, and 4 hours after the start of reperfusion from jugular veins and veins of the segment of the large colon that underwent ischemia and reperfusion. A sandwich ELISA was developed for detection of equine calprotectin. Serum calprotectin concentrations and values of blood gas, hematologic, and biochemical analysis variables were determined. Results—Large colon ischemia caused metabolic acidosis, a significant increase in lactate and potassium concentrations and creatine kinase activities, and a nonsignificant decrease in glucose concentrations in colonic venous blood samples. Values of these variables after reperfusion were similar to values before ischemia. Ischemia and reperfusion induced activation of an inflammatory response characterized by an increase in neutrophil cell turnover rate in jugular and colonic venous blood samples and calprotectin concentrations in colonic venous blood samples. Conclusions and Clinical Relevance—Results of this study suggested that large colon ischemia and reperfusion caused local and systemic inflammation in horses. Serum calprotectin concentration may be useful as a marker of this inflammatory response.

Objective: To evaluate in vitro effects of gemcitabine alone and in combination with carboplatin on canine transitional cell carcinoma (TCC) cell lines. Sample: In vitro cultures of 5 canine TCC cell lines. Procedures: Cells were treated with gemcitabine, carboplatin, or a combination of both at various concentrations. Cell proliferation was assessed via a fluorescence-based microplate cell proliferation assay. Cell cycle was evaluated via propidium iodide staining, and apoptosis was assessed by measurement of caspase 3 and 7 enzymatic activity. Synergy between gemcitabine and carboplatin was quantified via combination index analyses. Results: Treatment of 5 canine TCC cell lines with gemcitabine or carboplatin decreased cell proliferation, increased apoptosis, and induced cell cycle arrest. Cell cycle arrest and apoptosis were markedly increased when cell lines were treated with both gemcitabine and carboplatin simultaneously or sequentially. Order of administration during sequential treatment did not consistently affect cell proliferation results in TCC cell lines. When TCC cell lines were treated with gemcitabine and carboplatin in combination at therapeutically relevant concentrations (gemcitabine concentration, < 10μM; carboplatin concentration, < 250μM), a significant decrease in cell proliferation was observed, compared with cell proliferation following treatment with gemcitabine or carboplatin alone. In combination, the effects of gemcitabine and carboplatin were synergistic in 3 of 5 cell lines and additive in the other 2. Conclusions and Clinical Relevance: Gemcitabine had antitumor effects on canine TCC cells in vitro, and the combination of gemcitabine and carboplatin had synergistic activity at biologically achievable concentrations.

The purpose of the present research was to compare the enzyme activity of alkaline phosphatase (ALP), lactate dehydrogenase (LDH), α-amylase, α-manosidase, β-N-acetyloglucosaminidase, β-glucuronidase, and β-galactosidase in the cervical mucus of cows during spontaneous and induced estrus. Friesian cows (n = 106) were assigned to 4 groups: 1) no treatment; 2) progesterone releasing intervaginal device (PRID) for 12 days plus pregnant mare serum gonadotrophin (PMSG) at the removal of the PRID; 3) PGF2α2 doses 11 days apart; and 4) PRID for 7 days plus PGF2α 1 dose, 24 hours before removal of the PRID. Fourteen cows were excluded from the trial because of an inadequate quantity of cervical mucus collected or a lost PRID. The cows from the 3 induced estrus groups were artificially inseminated (AI) twice, while those with spontaneous estrus received only a single AI. Cervical mucus samples were collected from all cows 5 to 30 min before the first AI. The results are summarized as follows: 1) ALP and α-amylase activity for spontaneous estrus were similar to those for induced estrus; 2) LDH activity levels during spontaneous estrus were significantly lower (P < 0.001) than that in the P4 and P4+PGF2α induced estrus groups; and 3) glycosidases' activity was significantly lower (P < 0.001) in the spontaneous estrus group than that in the induced estrous groups. In conclusion, the activity of most enzymes in the cervical mucus of cows, in the present study, was significantly different between the spontaneous and the induced estrus groups.

Objective-To determine the effects of recombinant equine interleukin -1beta (reIL-1beta) and 4 anti-inflammatory compounds on the expression and activity of cyclooxygenase (COX)-2 in cultured equine chondrocytes. Sample Population-Articular cartilage from 9 young adult horses. Procedure-Reverse transcriptase-polymerase chain reaction methods were used to amplify a portion of equine COX-2 to prepare a cDNA probe. Northern blot analysis was used to quantify the expression of COX-2 in first-passage cultures of equine articular chondrocytes propagated in media containing dexamethasone (DEX), phenylbutazone (PBZ), polysulfated glycosaminoglycan, and hyaluronan, each at concentrations of 10 and 100 micrograms/ml and each with or without reIL-1beta. A commercial immunoassay was used to determine prostaglandin E2 (PGE2) concentrations in conditioned medium of similarly treated cells to quantify COX-2 activity. Results-Addition of reIL-1beta increased the expression of COX-2 in a dose-dependent manner, which was paralleled by an increased concentration of PGE2 in culture medium. Concentration of PGE2 in spent medium from reIL-1beta-treated chondrocytes was significantly reduced by DEX and PBZ; however, only DEX significantly reduced gene expression of COX-2. Conclusions and Clinical Relevance-Prostaglandin E2 is considered to be an important mediator in the pathophysiologic processes of arthritis, and cultured chondrocytes respond to interleukin-1 with enhanced expression and activity of COX-2. Palliative relief in affected horses is probably attributable, in part, to inhibition of PGE2 synthesis; however, analysis of these data suggests that of the 4 compounds tested, only DEX affects pretranslational regulation of the COX-2 gene in cultured equine chondrocytes.

Microcystin and related toxic peptides produced by cyanobacteria (blue-green algae) are potent and selective inhibitors of protein phosphatases 1 and 2A. We adapted existing enzymatic techniques to analyze the liver of rainbow trout after oral administration of hepatotoxic cyanobacteria. Liver tissue was removed 3 and 12 hours after treatment, and phosphatase activity was determined in liver extracts, using a specific phosphoprotein substrate. In all samples from fish exposed to toxic cyanobacteria, phosphatase activity was suppressed, whereas the control enzyme, lactate dehydrogenase, present in the same liver extract, was not affected by cyanobacteria. Thus, experimental poisoning by hepatotoxic cyanobacteria resulted in an abnormally low ratio of phosphatase to lactate dehydrogenase activity in the liver extracts. These results indicate that specific inhibition of phosphatases 1 and 2A may provide a useful diagnostic tool to determine the early effects of cyanobacteria toxic peptides directly in liver samples from poisoned animals. Although this test was developed with rainbow trout, it should be possible to extend the analysis of liver phosphatase activity to other species, including sheep and cattle, which are frequently affected by hepatotoxic cyanobacteria.

Methods available for measurement of plasma lipoprotein-cholesterol concentrations and activities of lipoprotein lipase, hepatic lipase, lecithin:cholesterol acyl transferase (LCAT), and cholesteryl ester transfer protein were adapted for use in cats. A combined ultracentrifugation/precipitation procedure was used to isolate very low-density lipoproteins (VLDL), then to separate low-density lipoproteins (LDL) from high-density lipoproteins (HDL). The reagent used, 92 mM heparin-manganese chloride, provided complete precipitation of LDL with only trace and insignificant contamination by HDL. Efforts to selectively measure lipoprotein lipase activity in plasma, collected after IV injection of heparin, by inhibiting hepatic lipase with sodium dodecyl sulfate were unsuccessful, and the activity of this enzyme was calculated as the difference between total and hepatic lipase activities. The latter was measured in the presence of high salt concentration to inhibit lipoprotein lipase. Cholesterol esterifying activity was identified in feline plasma and was typical of LCAT, in that it was dependent on apolipoprotein A-I as a cofactor. The intra-assay and interassay coefficients of variation for measurement of lipoprotein lipase, hepatic lipase, and LCAT activities were 18.4, 4.6, and 7.2%, and 20.4, 10.7, and 5.3%, respectively. Appreciable cholesteryl ester transfer protein activity was not detected in either undiluted or diluted plasma. These methods were subsequently used to investigate the effects of pregnancy and lactation on lipoprotein metabolism in a group of 10 queens. Plasma concentrations of cholesterol and triglycerides were unaltered during pregnancy, but the concentrations of VLDL-cholesterol increased and those of HDL-cholesterol decreased. During lactation, the concentrations of cholesterol and triglycerides decreased owing to reductions in VLDL-cholesterol and LDL-cholesterol concentrations and continued suppression of HDL-cholesterol. These changes were associated with alterations in the activities of lipoprotein lipase, which increased after parturition, and hepatic lipase, which increased during pregnancy and lactation, that may help explain their metabolic origins. The activity of LCAT remained unchanged.

Icterus condemnations compose a substantial proportion (41%) of total condemnations of bob veal, the class of veal composed of calves < 3 weeks old and weighing up to 68 kg. At postmortem examination, bob veal condemned because of icterus have generalized yellow discoloration of tissues, which is commonly associated with large, yellow liver (fatty liver), and a paucity of other gross pathologic changes. To establish that the generalized yellow discoloration was attributable to high tissue bilirubin concentrations and to examine the underlying mechanism(s) that might be responsible, blood samples and tissue specimens were obtained from clinically normal and icteric bob veal calves at slaughter. For comparison, blood samples were collected from clinically normal, 1- to 5-day-old Holstein calves being raised on local dairy farms. Hematologic and serum biochemical analyses were obtained for the 3 groups of calves (normal local, normal slaughter, and icteric slaughter), and tissues of slaughter calves were examined for histologic evidence of inflammatory or degenerative changes. Mean +/- SD total bilirubin concentration and creatine kinase (CK) activity in icteric bob veal (3.3 +/- 0.8 mg/dl; 869 +/- 788 U/L), normal bob veal (1.4 +/- 0.7 mg/dl; 486 +/- 890 U/L), and normal local calves (0.5 +/- 0.2 mg/dl; 156 +/- 158 U/L) were significantly different. When data for both normal and icteric bob veal calf groups were combined for analysis, total bilirubin concentration regressed significantly on hepatic lipid scores (P = 0.00003) and CK activity (P = 0.00049). Colostrum consumption was determined by measuring serum total protein concentration and serum gamma-glutamyltransferase activity. Bob veal calves that had not consumed colostrum had significantly higher total bilirubin (P = 0.00005) and CK (P = 0.0008) values. It was concluded that normal and icteric bob veal calves have significant increase in total bilirubin concentration, and icterus of bob veal calves is secondary to unconjugated hyperbilirubinemia. Lack of colostrum consumption was strongly correlated with icterus in bob veal calves.

Pharmacokinetic variables of skeletal muscle creatine kinase (CK) activity after IV administration of a muscle extract; CK bioavailability after IM administration of the muscle extract; and effect of IM administration of saline solution, to appreciate the possible release of CK consecutive to muscle puncture, were determined in 6 cows. A general equation for the quantitative estimation of skeletal muscle damage also was derived. Administration of saline solution IM had no effect on plasma CK activity (ANOVA, P > 0.05) in any of the cows. After IV administration of the muscle extract (150 U/kg of body weight), mean volume of the central compartment, plasma half-life, and plasma clearance of CK were 0.027 +/- 0.007 L/kg, 520 +/- 109 minutes, and 6.43 +/- 2.29 ml/kg/h, respectively. After IM administration (150 U/kg), mean bioavailability of CK was 51 +/- 17% and maximal plasma CK activity (500 +/- 97 U/L) was observed at 454 +/- 131 minutes. The rate of CK activity entry into plasma was determined by use of deconvolution analysis. Two peaks were observed; the first appeared before the 30th minute after IM administration, and the second appeared at 3.3 +/- 1.1 hours. Amplitudes were 6.31 +/- 4.45 and 6.57 +/- 3.08 U/kg/h, for the first and the second peaks, respectively. The quantity of CK liberated from control muscle was 0.69 +/- 0.12 U/kg/h, corresponding to a normal daily catabolism of 5.8 +/- 1.0 mg of muscle/kg. From these results, the following equation can be proposed to determine the corresponding mean equivalent of destroyed muscle (Qmuscle, test article) after IM administration of a test article: Qmuscle, test article (g/kg) = 4.41 X 10(-6) AUC (U/h/L), with AUC being the CK plasma activity area under the curve.