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.

The concentration of carbonic anhydrase III isoenzyme (cA-III) in serum samples from 216 clinically normal Thoroughbreds was determined by use of an enzyme immunoassay. The concentration range of cA-III was from 16.0 to 254.5 ng/ml (mean, 56.5 +/- 11.9 ng/ml). Significant differences were not detected according to age or sex. To confirm whether serum cA-III concentration was high in horses with muscle disease, serum samples of 11 horses with exertional rhabdomyolysis were analyzed by enzyme immunoassay. Their serum cA-III concentration was about 56 times (3,136 +/- 2,610 ng/ml) that of healthy Thoroughbreds. Concentration of cA-III was higher in horses with rhabdomyolysis that had been transiently recumbent than in horses with mild disease that were reluctant to move. Blood samples obtained serially from 6 horses with exertional rhabdomyolysis were studied. Serum activities of aldolase, creatine kinase, aspartate transaminase, and lactate dehydrogenase were high. Increases and decreases in concentration of cA-III were more rapid than that for aldolase, creatine kinase, aspartate transaminase, and lactate dehydrogenase activities; thus, cA-III may be clinically applicable as a diagnostic marker for muscle disease in horses.

Histomorphologic/morphometric evaluation, leukocyte scintigraphy, and myeloperoxidase activity were used to determine whether neutrophils accumulate in the large colon of horses during low-flow ischemia and reperfusion. Twenty-four adult horses were assigned to 1 of 3 groups: group 1, sham-operated (n = 6); group 2, 6 hours of ischemia (n = 9); and group 3, 3 hours of ischemia and 3 hours of reperfusion (n = 9). Low-flow ischemia of the large colon was induced in horses of groups 2 and 3 by reducing colonic arterial blood flow to 20% of baseline. Radiolabeled (99mTc) autogenous neutrophils were injected at 175 minutes, which corresponded to 5 minutes prior to reperfusion in group-3 horses. Full-thickness biopsy specimens of the left ventral colon were collected at baseline and at 30-minute intervals for 6 hours; a portion of the biopsy specimen was placed in formalin for histologic examination, and the remainder was used to measure mucosal radioactivity and myeloperoxidase activity. There were no differences in baseline mucosal neutrophil index, mucosal neutrophil numbers, submucosal venular neutrophil numbers, mucosal radioactivity, or mucosal myeloperoxidase activity among groups, or over time in group-1 horses. Neutrophils accumulated in the colonic mucosa during ischemia and further increased at reperfusion, as indicated by neutrophil index (morphology) and mucosal neutrophil numbers (morphometry); mucosal neutrophil index was significantly (P < 0.05) greater in group-3 horses during reperfusion than at the corresponding periods of ischemia in group-2 horses. Neutrophil numbers were significantly (P < 0.05) increased in submucosal venules at 10 minutes of reperfusion in group-3 horses and were significantly (P < 0.05) greater in group-3 than in group-2 horses during the interval from 3 to 6 hours. Mucosal radioactivity significantly (P < 0.05) increased at reperfusion in group-3 horses; there was a trend (P = 0.076) toward greater mucosal radioactivity in group-3, compared with group-2 horses, throughout the 3- to 6-hour interval. There were no differences in mucosal myeloperoxidase activity among or within any of the 3 groups over time. Neutrophils accumulated in the large colon of horses during low-flow ischemia and reperfusion. Neutrophil infiltration was detected by histologic examination and leukocyte scintigraphy, but not by measurement of myeloperoxidase activity. The accumulation of neutrophils during ischemia and the further neutrophil infiltration during reperfusion indicate that neutrophils may contribute to reperfusion injury of the large colon.

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.

High serum alkaline phosphatase (ALP) activity is considered a sensitive marker of cholestasis in most mammalian species, including dogs. Induction of high serum ALP activity in association with cholestasis is dependent on high hepatic bile acids concentrations. Treatment of dogs with glucocorticoids also results in high serum ALP activity. The possible causal relation between serum ALP activity and bile acids concentration was investigated in dogs treated with glucocorticoids. The relation of glucocorticoid treatment to changes in the activity of individual ALP isoenzymes, alanine transaminase (ALT) and gamma-glutamyltransferase (GGT) also was investigated. Eight conditioned dogs were given 4 mg of prednisone/kg of body weight, IM, daily for 10 days. Blood samples were taken prior to treatment and on treatment days 3, 5, 7, and 10. Liver tissue was then taken from each dog. Serum total ALP activity was significantly (P < 0.05) high at day 3 in prednisone-treated dogs. Isoenzyme analysis indicated that this increase was attributable to an increase in the liver ALP isoenzyme (LALP). Significant increases in serum corticosteroid-induced ALP (CALP) and bone ALP were first observed on days 7 and 10, respectively. Serum ALT and GGT activities were significantly increased by day 5. Increased serum or hepatic tissue bile acids concentrations were not observed in prednisone-treated dogs, compared with values in 8 clinically normal (control) dogs, but were high in 3 dogs with complete bile duct ligation. Hepatic activities of LALP, CALP, and GGT were higher in prednisone-treated dogs than values in controls, indicating probable increased hepatic synthesis of these enzymes. Hepatic ALT activity was not increased. The ratio of serum to tissue LALP activity was increased in prednisone-treated dogs, compared with values in controls, indicating that LALP may have been preferentially released into serum. There was no difference in the ratio of serum to liver GGT activity between prednisone-treated dogs and controls. The LALP and GGT ratios were increased in bile duct-obstruction dogs. It was concluded that, although LALP is the principal ALP isoenzyme in serum during the first 10 days of prednisone treatment, hepatic bile acid concentrations are not increased and, therefore, are not likely to be responsible for induction and release of ALP into serum. Prednisone may, therefore, be directly responsible for induction of ALP activity in dogs treated thusly.

To assess right colic artery blood flow and relevance of xanthine dehydrogenase/xanthine oxidase after experimentally induced strangulation obstruction and reperfusion of the colon, 5 ponies were subjected to 2.5 hours of complete ischemia of the left dorsal and ventral colons, allowed to recover from surgery, and monitored during a 48-hour reperfusion period. Five ponies were subjected to sham surgery and served as controls. All ponies had a Doppler ultrasound blood flow monitor implanted on the right colic artery near the pelvic flexure 10 to 14 days prior to the ischemic period. Colic artery blood flow was monitored prior to, during, and for 4 hours after surgery. Blood samples from the right colic artery and vein distal to the obstruction site were collected during surgery (prior to ischemia, after 1 and 2 hours of ischemia, and after 10 and 60 minutes of reperfusion) for determination of arterial and venous blood gas tensions and electrolytes. Prior to surgery, blood selenium and plasma vitamin E (alpha-tocopherol) concentrations and blood glutathione peroxidase (GPX) activity were determined to assess the status of endogenous antioxidants. Combined xanthine dehydrogenase (XDH) plus xanthine oxidase (XO) activity, and XO activity alone (nanomoles per minute per gram of tissue) were determined, using a dual-spectrophotometric technique. Xanthine dehydrogenase and oxidase activities were determined prior to ischemia, after 1 and 2 hours of ischemia, and at 1 and 48 hours after reperfusion. Median blood flow in the experimental and control groups (156 ml/min and 110 ml/min, respectively) was not statistically different before surgery, and was significantly (P < 0.02) lower in the experimental (4 ml/min) vs the control group (72.5 ml/min) during the ischemic period. Experimental ponies had significantly (P < 0.03) lower right colic artery blood flow during the 4 hours immediately after recovery from anesthesia. Significant difference was not observed in right colonic venous bicarbonate concentration between groups at any time. Median right colonic venous P(CO2), pH, and standard base excess were different (P < 0.001) between groups during the ischemic period only. Median venous oxygen saturation and median venous P(O2) were significantly (P < 0.001) lower in the experimental ponies at the end of 2 hours of ischemia, but were significantly (P < 0.05) increased during the reperfusion phase. Median venous potassium concentration was significantly (P < 0.01) higher in experimental ponies during the ischemic and reperfusion phases. Vitamin E and GPX values were within normal limits for all ponies. Median selenium concentration was < 15 microgram/dl; however, there were no significant differences between control and experimental ponies. Only 3 of 10 ponies had measurable XHH/XO activity at the beginning of the experiment. Enzyme activity was detected in 1 additional pony during the ischemic period. However, in all 4 ponies in which XDH/XO activity was detected, enzyme activity was low (10 to 36 nmol/min/g). On the basis of macroscopic and histologic examination of the large colon, evidence of reperfusion injury was not found in 4 of the 5 experimental ponies. The only pony with gross evidence of reperfusion injury did not have detectable XO activity. Results of the study indicate that hypoperfusion of the colon during the postischemic period may be a factor in deterioration of the colon observed clinically in equids with surgical correction of large-colon volvulus. Additionally, if reperfusion injury develops in the large colon, it probably is not mediated through the xanthine oxidase enzyme system: the activity of this enzyme in the large colon, when present, is negligible.

Foremilk, residual milk, and blood samples were studied for 10 days during acute mastitis episodes induced by endotoxin infused via the teat canal. Quarter milk and blood samples were collected frequently for 3 days after the infusion and thereafter once or twice daily. Leukocyte concentration in milk and blood was determined by flow cytometry. Within 2 hours after infusion of the endotoxin, clinical mastitis was observed. Total leukocyte concentration and proportion of neutrophils increased significantly (P < 0.05) by postinfusion hour (PIH) 2 in foremilk and by PIH 4 in residual milk. From PIH 2, serum albumin content and N-acetyl-beta-D-glucosaminidase activity were significantly increased in both fractions. Neutrophils were the predominant leukocyte population in both fractions until PIH 59. From PIH 72, lymphocytes were the predominant cell population until PIH 175 in foremilk and until PIH 223 in residual milk. Serum albumin content and N-acetyl-beta-D-glucosaminidase activity in residual milk was significantly lower than in foremilk from PIH 4 to 24 and from PIH 24 to 59, respectively. Regarding total and differential leukocyte counts, values for the 2 fractions followed the same pattern throughout the course of inflammation, probably owing to frequent sample collection. Total and differential cell counts tended to differ between the fractions during some periods, although differences were not statistically significant. When samples were taken less frequently, the total leukocyte concentration in residual milk was higher than that in foremilk. Although sample collections were frequent, clustering of immature neutrophils was not observed in the cytofluorogram of blood leukocytes in this study. Residual milk seems to be the fraction that best reflects the condition in the quarter at the particular time when the milk sample is taken. Results also indicate that residual milk reflects the condition of the secretory tissue, as well as the lower regions of the gland.

An epidemiologic study of Pasteurella haemolytica serovar 1 (Ph1) in market-stressed feeder calves from 7 farms in eastern Tennessee was conducted. The nasal mucus of each calf was cultured sequentially at the farm of origin (day 0), at an auction market (day 133), and at a feedyard in Texas (days 141, 148, 155, and 169). Of the 103 calves tested, 77 were culture-positive, including 1 on day 0, 1 on day 133, 20 on day 141, 57 on day 148, 50 on day 155, and 14 on day 169. From the 143 Ph1 isolates, 20 enzyme profiles were determined by use of a commercial enzyme system that detects 19 enzymatic reactions; 4 antimicrobial susceptibility profiles were obtained, using the disk-diffusion method, which evaluated susceptibility to 11 antibacterial drugs. All isolates were positive for acid phosphatase and alkaline phosphatase, but were negative for alpha-galactosidase, alpha-mannosidase, beta-glucosidase, beta-glucuronidase, cystine aminopeptidase, N-acetyl-beta-glucosaminidase, and trypsin. Other positive enzyme reactions included: leucine aminopeptidase, 140 Ph1 isolates; phosphohydrolase, 90 isolates; alpha-fucosidase, 63 isolates; esterase (C4), 59 isolates; valine aminopeptidase, 30 isolates; esterase lipase (C8), 24 isolates; beta-galactosidase, 2 isolates; and alpha-glucosidase, chymotrypsin and lipase (C14), 1 isolate each. Thirty-four Ph1 profiles were identified, using combined enzyme and antimicrobial susceptibility profiles. The data indicate that the strains isolated during the feedyard period may have been determined more by farm of origin (P < 0.001) than by habitation with calves from other farms while in the feedyard. The combined enzyme and antimicrobial susceptibility profile method is a rapid and simple epidemiologic technique for tracking Ph1 strains in market-stressed feeder calves.

Acyloxyacyl hydrolase (AOAH) is a lysosomal enzyme found in neutrophils and macrophages that acts to partially deacylate the lipid-A component of the endotoxin of gram-negative bacteria rendering it less toxic, yet maintaining much of its immunostimulatory potential. We have found that the activity of neutrophil AOAH per cell increased during localized inflammation. The purpose of this study was to determine the mechanism(s) responsible for these increases in neutrophil AOAH activity. Because changes in neutrophil maturity commonly are associated with inflammation, intravascular infusion of purified gram-negative bacterial lipopolysaccharide and SC injection of bovine recombinant granulocyte colony-stimulating factor was used to induce large numbers of circulating immature neutrophils. Immature neutrophils were found to have AOAH activity equal to that of mature cells; however, when neutrophils were stimulated in vitro with known activators, AOAH activity of activated cells was more than that of unstimulated cells. The increase in AOAH activity was inversely related to prestimulation activity. Increases in AOAH activity after neutrophil activation were not a result of de novo synthesis of the enzyme, because cycloheximide did not prevent activation-induced increases in activity.

A selected group of pharmaceutical compounds were evaluated for the ability to inhibit the biochemical activity of fibrinoligase (coagulation factor XIIIa) in pooled equine plasma. Criteria for the pharmaceuticals selected were based on the mechanism of the transglutamination biochemical reaction mediated by coagulation factor XIIa . These criteria were complemented by recognition of the molecular configuration and chemical composition of amino acid residue side chains involved in the process of covalent fibrin monomer polymerization (cross-linking, transglutamination) mediated by this enzyme. Each pharmaceutical was evaluated individually and in combination with other potential coagulation factor XIIIa inhibitors in an effort to detect additive and synergistic phenomenon. In this context, pharmaceuticals with a carbonylamide (eg, cefuroxime, Girard's reagent-P, prolinamide) were applied in concert with compounds with a terminal amine (eg, D-arginine, L-lysine) functional group. In concept, this method theoretically served to competitively simulate glutamine and lysine amino acid residues within strands of fibrin monomer substrate involved in phase I (carbonylamide) and phase II (terminal amine) of the transglutamination reaction (covalent fibrin monomer cross-linking). Halogen-dinitro and ethylene compounds were also evaluated because of their reported ability to inactivate enzyme systems dependent on an intact sulfhydryl group located at their biochemically active site (eg, cystine amino acid residue). This group of pharmaceutical compounds failed to inhibit the biochemical activity mediated by coagulation factor XIIIa in equine plasma.