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.

Indices of renal function and damage were measured in 12 healthy male adult llamas fed a diet of mixed alfalfa/grass hay (mixed hay) and water ad libitum. Using a collection bag fitted over the preputial area, urine samples were collected at 6, 12, and 24 hours. Serum samples were obtained concurrently to determine endogenous creatinine clearance (CL), total (TE) and fractional excretion (FE) of electrolytes (Na, K, Cl, P), electrolyte CL, urine and serum osmolality, urine enzyme activities (gamma-glutamyltransferase and N-acetyl-beta-D-glucosaminidase), and urine protein concentration. Urine production was quantified. Three months later, 10 of the 12 llamas were fed a grass hay diet and water ad libitum. Similar samples were obtained, and similar measurements were made. Urine production was higher when the llamas were fed the mixed hay diet. Total urine volume for llamas fed mixed hay ranged from 628 to 1,760 ml/24 h, with a median of 1,307.5 ml/24 h, compared with a range of 620 to 1,380 ml/24 h and a median of 927.50 ml/24 h for llamas fed grass hay. Median urine osmolality was higher in llamas fed mixed hay (1,906 mOsm/kg of body weight, with a range of 1,237 to 2,529 mOsm/kg), compared with llamas fed grass hay (1,666 mOsm/kg with a range of 1,163 to 2,044 mOsm/kg). Creatinine CL did not vary significantly over time for either diet. Median creatinine CL was higher for llamas fed mixed hay, compared with llamas fed grass hay--0.78 ml/min/kg with a range of 0.20 to 1.83 ml/min/kg vs 0.45 ml/min/kg with a range of 0.13 to 3.17 ml/min/kg. Clearances for K and Cl varied significantly among the periods. However, median CL for Na and P did not vary over time for either diet. Overall values for these electrolytes in llamas fed mixed hay and grass hay diets were: CL(Na), 0.001 and 0.002 ml/min/kg and CL(P), 0.0006 and 0.0004 ml/min/kg respectively. The FE rates of K, Cl, and P did not vary significantly over time for either diet. Median respective FE for these electrolytes in the llamas fed mixed hay and grass hay diets include: FE(K), 84.90 and 63.10%; FE(Cl), 0.85 and 1.30%; and FE(P), 0.10 and 0.10%. Fractional excretion of Na varied over time for both diets and could not be expressed accurately as an overall median. Median respective TE of electrolytes for llamas fed the mixed hay and grass hay diets were: TE(Na), 0.007 and 0.03 mEq/kg/h; TE(Cl), 0.04 and 0.06 mEq/kg/h; and TE(P), 0.0002 and 0.00 mg/kg/h; TE(K) varied significantly (P < 0.05) over time for both diets. Urine gamma-glutamyltransferase activity changed significantly (P < 0.05) over time. Urine N-acetyl-beta-D-glucosaminidase activity was influenced by an interaction between diet and time. Median urine protein concentration was 26.0 mg/dl, with a range of 11.0 to 73.0 mg/dl for llamas fed mixed hay, and was 28.0 mg/dl, with a range of 16.0 to 124.0 mg/dl for llamas fed grass hay.

Dogs are particularly susceptible to development of glucocorticoid-induced hepatopathy, but the mechanisms are not well understood. We investigated the pathogenesis of glucocorticoid hepatopathy by examining sequential morphologic and biochemical changes in the liver of dogs during steroid administration. Six adult Beagles were given prednisolone acetate (4 mg/kg of body weight, once daily for 24 days, IM). Serum samples and percutaneous liver biopsy specimens were obtained before the start of the study (treatment day [TD] 0) and at TD 5, 10, 15, and 25. There were significant (P < 0.05) and progressive increases in serum activities of alkaline phosphatase, gamma-glutamyltransferase, and alanine transaminase. Light microscopic changes in liver biopsy specimens included progressive hepatocellular swelling and vacuolation. Electron microscopy revealed glycogen accumulation, peripheral displacement of organelles, and prominent dilatation of bile canaliculi, compared with findings at TD 0. Liver biopsy specimens taken at TD 25 had significantly (P < 0.05) increased activities of the plasma membrane enzymes, alkaline phosphatase and y-glutamyltransferase, and 5'-nucleotidase was significantly (P < 0.01) decreased. Subcellular fractionation on reorientating sucrose density gradients revealed high-density peaks of alkaline phosphatase and gamma-glutamyltransferase, compatible with a specific increase in the biliary canalicular component of the enzyme activities. Neutral gamma-glucosidase activity was shifted to the denser fractions, indicative of an increase in the proportion of rough to smooth endoplasmic reticulum and consistent with enhanced synthesis of plasma membrane proteins. There also was evidence for progressive increase in fragility of intracellular organelles, particularly lysosomes. These findings indicate that glucocorticoid he particularly in dogs is associated with progressive alterations not only to the plasma membrane, but also to other subcellular organelles.

We evaluated the pharmacokinetics of IV administered sodium heparin and the pharmacodynamic effect of heparin on lipoprotein lipase (LPL) activity Horses were allotted to 3 groups. Plasma samples were obtained from each horse before and at various times for 6 hours after heparin administration for determination of heparin concentration, LPL activity, and activated partial thromboplastin time (APTT). The disposition of heparin was dose dependent. The area under the plasma heparin concentration vs time curve (AUC) increased more than proportionally with dose, indicating that heparin elimination was nonlinear. Total clearance of heparin was similar after the 40 and 80 IU/kg of body weight dosages, averaging 0.45 and 0.36 IU/kg/min, respectively. However, after administration of the 120 IU/kg dose, clearance was significantly less than that after the 40 IU/kg dose. The half-life of heparin averaged 53, 70, and 136 minutes after 40, 80, and 120 IU/kg, respectively, with significant differences observed between the low and high doses. In contrast to heparin, the area under the plasma concentration vs time curve for LPL activity increased less than proportionally with dose. Maximal LPL activity observed was independent of dose, averaging 4.8 micromole of free fatty acids/ml/h. The APTT was significantly prolonged for 120 minutes after administration of the 40 IU/kg dose. Correlation coefficients for LPL activity vs either plasma heparin concentration or APTT were less than 0.7, indicating that neither laboratory measure can be used to accurately predict plasma LPL activity.

To examine the postnatal development of equine small intestine, biopsy specimens of jejunal mucosa from 8 ponies, between 6 and 28 weeks old, were subjected to analytical subcellular fractionation and assay of organelle marker enzymes. Fractionation revealed a reduction in the particulate brush border component of beta-galactosidase (lactase) activity between 6 and 28 weeks, and a corresponding increase in soluble activity, although the reduction in mean specific activity was not significant. There also was a decrease in the proportion of brush border to soluble aminopeptidase N activity, a relative loss of brush border gamma-glutamyltransferase activity, and a considerable decrease in the specific activity of alkaline phosphatase throughout the gradient fractions. In contrast, there were marked increases in activities of (alpha-glucosidase (maltase) and sucrase in the older ponies, accompanied by considerable changes in the intracellular distribution of particulate alpha-glucosidase activity, which was predominantly associated with endoplasmic reticulum at 6 weeks, whereas the large increase in activity observed by 28 weeks was clearly associated with the brush border. The modal density of brush borders also increased with age, suggestive of an increase in the glycoprotein-to-lipid ratio of the microvillar membrane. In contrast to these brush border changes, there was relatively little alteration in the activities or density distributions of marker enzymes for endoplasmic reticulum, basolateral membranes, mitochondria, or lysosomes. These findings indicate that maturation of equine intestinal epithelium during the first few months of life results in major changes in the properties and enzyme composition of enterocyte brush borders.

Activities of diagnostically important enzymes were measured in serum and lysates of liver, kidney, skeletal muscle, heart, intestine, lung, and pancreatic tissues from wild-caught yellow rat snakes, Elaphe obsoleta quadrivitatta. All samples were analyzed for alkaline phosphatase, lactate dehydrogenase (LD), aspartate transaminase (AST), alanine transaminase, gamma-glutamyltransferase, and creatine kinase (CK) activities. The major enzyme activities found in the liver were LD and AST. The kidney had moderate activities of LD, AST, alanine transaminase, and CK. Skeletal muscle and heart contained high CK activity. Intestine, lung, and pancreas had low activities for most enzymes analyzed. Little to no gamma-glutamyltransferase activity was found in serum or tissues analyzed. Serum enzyme activities in yellow rat snakes were similar to those described for other reptile species, except for serum CK activity, which was increased in rat snakes.

Serum alpha-mannosidase activity and swainsonine concentration were determined in 4 cattle and 15 sheep (3 groups of 5 each) that were administered ground locoweed (Oxytropis sericea or Astragalus lentiginosus) containing swainsonine at dosages of approximately 0.8 mg/kg of body weight/d (cows, 30 days each) and 0, 1.0, and 1.5 mg/kg/d (sheep, 11 days each). The cattle developed mild clinical signs of locoism, including signs of depression, lethargy, and slight intention tremors. Clinical signs of toxicosis were not observed in the sheep. Within 24 hours of initial treatment, serum alpha-mannosidase activity in cows and sheep, measured by the release of 4-methylumbelliferone from an artificial substrate, was markedly decreased to 28 and 40 micromoles of 4-methylumbelliferone/L, respectively. Mean serum alpha-mannosidase activity of control cows and sheep was 400 +/- 94 and 422 +/- 42 (mean +/- SD), respectively. In the treated animals, decreased serum alpha-mannosidase activities returned to normal or higher activities within 6 days after treatment was discontinued. Using a jack bean alpha-mannosidase assay, increased swainsonine activity (153, 209, and 381 ng/ml, respectively) was detected in the serum of cattle and of sheep in the high- and low-dose groups within 24 hours after treatment with locoweed. Swainsonine concentration remained high, with mean concentrations of 204, 432, and 395 ng/ml (cows and 2 sheep groups, respectively) during the treatment period. After treatment, swainsonine was rapidly cleared, with estimated serum half-life of 16.4, 17.6, and 20.3 hours (cows, and high- and low-dose sheep groups, respectively). Significant differences in either alpha-mannosidase activity or swainsonine concentration were not detected between the 2 groups of treated sheep. These results suggest that serum alpha-mannosidase and swainsonine values are sensitive indicators of locoweed intoxication in cattle and sheep. Furthermore, it suggests that swainsonine is rapidly absorbed, resulting in rapid inhibition of serum alpha-mannosidase activity, leading to high serum swainsonine concentration. After exposure is eliminated, swainsonine is rapidly cleared from the serum, with serum alpha-mannosidase activity returning to normal values shortly thereafter.