Development of an enzyme-linked immunosorbent assay for equine neutrophil elastase measurement in blood: preliminary application to colic cases.

Equine neutrophil elastase (NE) is a protease released in inflammatory diseases and participating in tissue destruction. To measure NE in horse plasma to assess its role in pathological conditions, we purified elastase from equine neutrophils by a double step chromatography and obtained a pure protein of 27 kDa, 4 kDa smaller than the NE 2A previously purified (Scudamore et al., 1993; Dagleish et al., 1999), which was likely to be NE 2B. We developed an ELISA by using two specific polyclonal antibodies obtained from rabbit and guinea pig. The sandwich complex was detected using a secondary antibody conjugated to alkaline phosphatase. The ELISA showed good precision and accuracy, with intra- and inter-assay coefficients of variation below 10% for equine NE concentrations ranging from 1.875 to 60 ng/ml. A stable plasma NE value, unaffected by the delay of centrifugation (over 4h), was obtained with plasma from EDTA anticoagulated blood. The mean value (+/-SEM) measured in 37 healthy horses was 32.53+/-4.6 ng/ml. NE level in plasma of horses with colic at the time of admission was significantly higher than in healthy horses. Our results indicate that the ELISA technique we developed to measure plasmatic NE is a powerful tool for studying the role of elastase in equine inflammatory disease. In future, the application will be extended to other equine biological fluids.

Myeloperoxidase assay in plasma and peritoneal fluid of horses with gastrointestinal disease.

Gastrointestinal disorders, especially strangulating intestinal obstructions, are still a major cause of illness and death in the horse. Circulating lipopolysaccharides may activate both neutrophils and monocytes. The activated neutrophils release myeloperoxidase (MPO), a specific enzyme with strong oxidative activity. The aim of this study was to evaluate MPO concentrations in the plasma and peritoneal fluid (PF) of horses with colic and to check the hypothesis that these concentrations would be higher in a case of strangulating obstruction than in cases of nonstrangulating disease. By using a specific enzyme-linked immunosorbent assay for equine MPO, we determined the MPO concentrations in horses admitted to a clinic for colic. Horses with nonstrangulating or strangulating obstruction of the large intestine (NSLI or SLI), strangulating obstruction of the small intestine (SSI), or inflammatory bowel disease (IBD) were compared with healthy horses. The horses with SLI, SSI, or IBD had significantly higher MPO levels in plasma and PF than did those in the other 2 groups. The mean plasma level was significantly higher in the horses with NSLI than in the healthy horses. High MPO values in PF indicated necrotic bowel. These results show that neutrophil activation occurs during nonstrangulating and strangulating intestinal obstruction in horses and that the plasma and PF MPO concentrations may be a marker of the severity of the disease.

Measurements of blood flow and xanthine oxidase activity during postischemic reperfusion of the large colon of ponies.

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 PCO2, pH, and standard base excess were different (P < 0.001) between groups during the ischemic period only. Median venous oxygen saturation and median venous PO2 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.(ABSTRACT TRUNCATED AT 400 WORDS)

Observations on the isoenzymes of creatine kinase in equine serum and tissues.

The isoenzymes of creatine kinase have been measured in serum and selected tissues from horses. The distribution followed that reported in other species in that the MM dimer of the isoenzyme was present in voluntary and non-voluntary muscle, thyroid, liver, spleen, lung and intestine. The BB dimer of the isoenzyme was predominant in brain, pancreas, kidney, intestine, lung, spleen, liver and thyroid. In contrast, in 4 hearts examined less than 1.5 per cent of the total creatine kinase activity was attributable to the MB form of the isoenzyme. The MB isoenzyme was, however, present in intestine and spleen. In this series the isoenzymes were separated by ion exchange chromatography and their presence confirmed by cellulose acetate electrophoresis. It was concluded that as insignificant quantities of MB isoenzyme are present in equine heart, measurement of this isoenzyme in serum cannot be used to detect myocardial or cardiac damage in the horse. Further detailed studies need to be undertaken to assess the clinical relevance of the isoenzyme pattern in serum or other body fluids, if the measurement of creatine kinase isoenzymes in serum is to be used as a diagnostic aid.

Origin an importance of increased alkaline phosphatase activity in peritoneal fluids of horses with colic.

The origin of increased alkaline phosphatase (ALP) activity in peritoneal fluid (PF) of horses with clinical signs of abdominal pain was investigated to determine the usefulness of measuring ALP in PF in the diagnosis of small intestinal injury. The ALP isoenzymes in PF from 10 clinically normal horses and from 50 horses with clinical signs of acute abdominal pain were analyzed for their sensitivities to inhibition by L-phenylalanine, L-homoarginine, and levamisole and to inactivation by heat (56 C, 15 minutes). The enzymes also were discriminated by their patterns of migration during polyacrylamide gel disc electrophoresis. Of 50 horses with colic, 20 had ALP activity in PF at least 3 times the upper limit of normal. Of these 20 horses, 10 had marked increases of ALP activity in PF ranging from 10 to 150 times the mean value of activity as determined in the 10 normal horses. In the 50 horses with colic, ALP values in serum were within the normal range. In 19 of the 20 sick horses, the ALP in PF had properties different from small intestinal ALP. Of the 10 PF samples with markedly increased ALP activity, 9 had a group of properties that were unique for granulocytic ALP. The clinical diagnoses for the 10 horses with markedly increased ALP activity in PF included thromboembolic colic (4 horses), colonic torsion (2 horses), small intestinal volvulus (2 horses), peritonitis (1 horse), and salmonellosis (1 horse). Properties of the enzyme in the 10 PF samples with moderately increased ALP activity were compatible with a granulocytic origin, but insufficient enzyme concentration precluded electrophoretic confirmation of the source. The PF from 1 horse had a mixture of ALP isoenzymes derived from granulocytes and small intestinal mucosa. Of the 50 horses with colic, 6 had severe small intestinal disease without increased ALP activity in PF. Apparently, increased ALP activity in PF cannot be used as a reliable indicator of small intestinal injury in horses, because the ALP is predominantly granulocytic in origin.

Affinity labeling of the Escherichia coli aspartate-beta-semialdehyde dehydrogenase with an alkylating coenzyme analogue. Half-site reactivity and competition with the substrate alkylating analogue.

3-Chloroacetylpyridine-adenine dinucleotide phosphate (clac3PdADP+, a NADP+ alkylating analogue, irreversibly inactivates aspartate-beta-semialdehyde dehydrogenase with pseudo-first-order kinetics. NADP+ and NADPH, but not the substrate, protected the enzyme from inactivation. The pH dependence of the inactivation kinetics was determined. Incorporation of 1 mol cl[14C]-ac3PdADP+/dimer totally inactivates the enzyme. Successive alkylation by the coenzyme analogue and by the substrate analogue, L-2-amino-4-oxo-5-chloropentanoic acid, was studied. After inactivation with the coenzyme analogue, no incorporation of the substrate analogue was detected. However, when the enzyme was first inactivated with the substrate analogue, the protein could subsequently be alkylated with the coenzyme analogue. The binding of NADP+ and NADPH to aspartate-beta-semialdehyde dehydrogenase was determined by fluorescence.