Characterization of total adenosine deaminase activity (ADA) and its isoenzymes in saliva and serum in health and inflammatory conditions in four different species: an analytical and clinical validation pilot study.

BACKGROUND: Measurement of adenosine deaminase (ADA) can provide information about cell-mediated immunity. This report's objective was to study the enzymatic activity of total ADA (tADA) and its isoenzymes ADA1 and ADA2 in canine, equine, porcine, and bovine serum and saliva and their changes in different inflammatory situations in each species. Besides, an automated method for ADA2 measurement was developed and validated. RESULTS: tADA was present in serum and saliva of healthy animals of the four species. Erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) concentration of 0.47 mM was needed for ADA1 inhibition in canine and porcine samples (serum and saliva) and bovine saliva, whereas for equine saliva 0.94 mM was needed. ADA2 activity was not detected in bovine serum and was very low or absent in equine serum and bovine saliva. An automated procedure to measure ADA2 consisting of adding EHNA to a commercial reagent for tADA measurement provided repetitive (coefficients of variation < 8.8% in serum and < 10% in saliva) and accurate (linearity of serial sample dilutions with R2 > 0.90) results, being equivalent to a manual incubation of the sample with EHNA at a similar concentration. Salivary tADA, as well as ADA1 and ADA2, were higher in dogs with leishmaniosis, horses with acute abdominal disease and pigs with lameness than in healthy animals. tADA and isoenzymes in saliva showed a positive significant correlation with serum ferritin in dogs (r = 0.602, P < 0.01; r = 0.555, P < 0.05; and r = 0.632, P < 0.01; respectively for tADA, ADA1 and ADA2) and serum C-reactive protein in pigs (r = 0.700, P < 0.01, for both tADA and ADA1; r = 0.770, P < 0.001, for ADA2), whereas salivary ADA2 significantly correlated with serum amyloid A in horses (r = 0.649, P < 0.01). In cows, salivary tADA and ADA1 significantly increased after calving, correlating with total white blood cell count (r = 0.487, P < 0.05, for both tADA and ADA1). CONCLUSIONS: The activity of total ADA and its different isoenzymes, can be measured in serum and saliva of dogs, horses, pigs and cows by a simple and fast procedure described in this report. When measured in saliva, these analytes correlated with other biomarkers of inflammation and it could potentially be used as a biomarkers of inflammation and immune activation in the species of this study.

The chlorinated AHR ligand 3,3',4,4',5-pentachlorobiphenyl (PCB126) promotes reactive oxygen species (ROS) production during embryonic development in the killifish (Fundulus heteroclitus).

Exposure to dioxin-like chemicals that activate the aryl hydrocarbon receptor (AHR) can result in increased cellular and tissue production of reactive oxygen species (ROS). Little is known of these effects during early fish development. We used the fish model, Fundulus heteroclitus, to determine if the AHR ligand and pro-oxidant 3,3',4,4',5-pentachlorobiphenyl (PCB126) can increase ROS production during killifish development, and to test a novel method for measuring ROS non-invasively in a living organism. The superoxide-sensitive fluorescent dye, dihydroethidium (DHE), was used to detect in ovo ROS production microscopically in developing killifish exposed to PCB126 or vehicle. Both in ovo CYP1A activity (ethoxyresorufin-o-deethylase, EROD) and in ovo ROS were induced by PCB126. In ovo CYP1A activity was inducible by PCB126 concentrations as low as 0.003 nM, with maximal induction occurring at 0.3 nM PCB126. These PCB126 concentrations also significantly increased in ovo ROS production in embryonic liver, ROS being detectable as early as 5 days post-fertilization. These data demonstrate that the pro-oxidant and CYP1A inducer, PCB126, increases both CYP1A activity and ROS production in developing killifish embryos. The superoxide detection assay (SoDA) described in this paper provides a semi-quantitative, easily measured, early indicator of altered ROS production that can be used in conjunction with simultaneous in ovo measurements of CYP1A activity and embryo development to explore functional relationships among biochemical, physiological and developmental responses to AHR ligands.

Practical Interpretation and Application of Exocrine Pancreatic Testing in Small Animals.

The pancreas remains a difficult organ to evaluate using laboratory methods alone. No single laboratory test is diagnostic of pancreatitis (chronic or acute) without other diagnostic modalities concurring with the diagnosis or ruling out other diseases. The diagnosis of pancreatitis is particularly difficult in cats, and pancreatitis often occurs with other diseases. The use of pancreatic cytology may be useful in diagnosing both inflammation and neoplasia. Exocrine pancreatic insufficiency (EPI) can be relatively easily diagnosed when clinically manifested by the measurement of trypsinlike immunoreactivity. Diagnosis is more difficult when EPI is subclinical.

Use of bovine myeloperoxidase as an indicator of mastitis in dairy cattle.

Bovine myeloperoxidase (MPO) is a heme protein consisting of both large and small polypeptide subunits. In mammals the role of MPO in defending against microbes is well documented. To evaluate the potential of using MPO in the diagnosis of udder infections in dairy cattle we developed a specific enzyme immunoassay for bovine MPO in milk. Antibodies against bovine MPO were produced using the purified enzyme. The ELISA utilizes two specific antibodies: one that is anti-MPO monoclonal and one that is anti-MPO polyclonal. For a total of 141 milk samples the correlation coefficient between the somatic cell count and MPO concentrations determined using the ELISA was 0.91. The ELISA showed good precision and accuracy in measuring MPO in milk, with a total variation of ca. 10%. The recoveries of known amounts of MPO from milk were satisfactory. Thus the stability of the enzyme in milk was judged to be good. Microorganisms were isolated in ca. 85% of the milk samples with elevated concentrations of MPO. Microorganisms were not isolated from more than 90% of non mastitic milk samples with low somatic cell counts where MPO was not detectable using ELISA. The results clearly show that the quantitative analysis of the amount of MPO in mastitic milk can be used to detect intramammary infections in dairy cattle.

Evaluation of abomasal enzyme and hormone levels in the diagnosis of ostertagiasis.

Pathophysiological changes in the ruminant abomasum caused by Ostertagia infections include changes in the activity and concentration of gastrointestinal enzymes and hormones. Under certain circumstances, increases in concentration also occur in the bloodstream and, as such, are detectable. Determination of serum pepsinogen levels is useful in evaluating the risk or presence of ostertagiasis Type I in a herd. It seems less reliable when used to diagnose (pre) ostertagiasis in individual animals. Measurement of the concentration of other zymogens is not useful. The variations in methodology to determine pepsinogen levels (e.g. biochemical and immunological measurements) are discussed. Serum gastrin levels are, generally, increased in animals with ostertagiasis. At present, gastrin is mainly determined by RIA assays using human gastrin antibodies, but few baseline data are available on normal levels in ruminants. The use of gastrin determination as a diagnostic tool in Ostertagia-infected ruminants is limited.

The use of selected plasma enzyme activities for the diagnosis of fatty liver-hemorrhagic syndrome in laying hens.

Profiles of plasma enzymes were compared in two strains of single comb white leghorn laying hens, a normal commercial strain and strain UCD-003, which is highly susceptible to fatty liver-hemorrhagic syndrome. Plasma activity of lactate dehydrogenase (LDH), glutamate dehydrogenase (GDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and creatine kinase (CK) averaged 194 +/- 27, 4.0 +/- 2.8, 146 +/- 20, 1.0 +/- 1.0, and 1041 +/- 268 U/liter, respectively in normal birds. Activities of LDH, GDH, AST, and ALT, but not CK, were significantly higher in UCD-003 than in normal hens. A bimodal distribution of activities of all enzymes was found in the UCD-003 hens, with some birds showing activities comparable with those of the normal hens and others with values that were 2-10 times greater than those found in normal hens. These results are consistent with the extensive hepatic lesions observed in the UCD-003 strain of birds. Average gross hemorrhagic scores from visual inspection (scale of 0-3) were 0.28 +/- 0.45 in normal birds and 1.63 +/- 0.94 in the UCD-003 birds. Even though no clear relationship was found between plasma enzyme activities and the extent of liver hemorrhage in individual birds, the UCD-003 hens consistently had average values significantly higher for plasma enzymes that indicate liver damage. The results suggest that measurement of enzyme activities indicative of liver damage in birds, particularly AST, LDH, and GDH, is a valuable tool in the diagnosis of fatty liver-hemorrhagic syndrome in a flock of layers.

Clinical chemical constituents in relation to liver amyloidosis in serum-producing horses.

Serum activities of gamma-glutamyl transferase (GGT), alkaline phosphatase (AP), aspartate aminotransferase (AST), and concentrations of total bilirubin and total bile acids were screened during a 5 year period in 27 horses used for production of hyperimmune serum. The horses investigated were regularly immunized with live cultures of the endotoxin-releasing bacteria Escherichia coli or Pasteurella multocida, the individual animals having undergone such treatment for periods varying from 2 weeks to 10 years. In a majority of the horses, GGT-activity had increased within 6 to 7 years of first having undergone immunization. Constantly high values seemed to co-incide with the presence of advanced liver amyloidosis, as demonstrated by histopathological examination after slaughter. The AP activity was also increased but only moderately compared with GGT. Individual values more than 10-fold greater than the upper reference limit were recorded for GGT, while the highest AP values were less than double the upper reference limit. Activity of AST and bilirubin concentrations remained unaffected, whereas the concentration of total bile acids rose after 6 to 7 years of immunization compared to the baseline value. It is concluded that the determination of serum activities of GGT may serve as a practical routine test for the evaluation of liver amyloidosis status in serum horses.

Creatine kinase in dog plasma: preanalytical factors of variation, reference values and diagnostic significance.

In the dog, plasma creatine kinase (CK) activity was stable up to one week at +4 degrees C and one month at -20 degrees C. Activity was higher in serum than in plasma due to interference by CK from the platelets. The reference values were determined in 232 dogs using the IFCC recommended method. There was a significant decrease in activity with age but no effect of sex. In adults, plasma CK exhibited a log-normal distribution ranging from 20 to 104 U per litre. In 510 dogs with various diseases, the overall sensitivity and specificity of CK determination were 40 per cent and 98 per cent, respectively. The numerous false negatives could result from the relatively short half-life of the enzyme, while the false positives could be due to secondary muscle damage.

Modified granulocyte test for determination of mannosidosis genotype of cattle.

Recovery of alpha-mannosidase, beta-N-acetylglucosaminidase and arylsulphatase activity from granulocytes is increased by inclusion of 154 mM sodium chloride, in place of 1 mM zinc chloride, in aqueous Triton X-100 used to extract the cells. Calculation of the ratio of alpha-mannosidase to beta-N-acetylglucosaminidase and to arylsulphatase permits diagnosis of the mannosidosis genotype without recourse to examination of smears of the granulocyte preparations.

Use of erythrocyte copper:zinc superoxide dismutase activity and hair or fleece copper concentrations in the diagnosis of hypocuprosis in ruminants.

Changes in the activity of superoxide dismutase, a copper-containing enzyme in erythrocytes (ESOD), and changes in copper in hair or fleece were compared with the changes in plasma copper during copper depletion and repletion in steers, lambs and ewes. During depletion the decline in ESOD began later than the decline in plasma copper: the lag varied from 0 to 80 days in individual steers and it was least evident when young rapidly growing lambs were subjected to severe depletion. ESOD activity eventually declined at only one-third to one-seventh of the rate shown by plasma copper, the difference being particularly marked in severely depleted lambs. Repletion of ESOD began after repletion in plasma copper and continued more slowly particularly in growing animals. Hair and fleece copper also responded relatively slowly to depletion and repletion. Low values of ESOD and hair or fleece copper may have diagnostic significance, indicating a more prolonged or intense deficiency of copper and a higher probability of clinical and production responses to copper therapy than low plasma or liver copper values.

Examination of milk samples from experimentally infected heifers by an N-acetyl-beta-D-glucosaminidase test and an antigen-capture ELISA.

Milk samples collected from normal and experimentally infected quarters of five heifers throughout their first lactation were examined by bacterial culture, milk cell count (MCC), N-acetyl-beta-D-glucosaminidase (NAGase) test and a monoclonal antibody based antigen-capture ELISA. The results were analysed according to the presence (mastitis positive) or absence (mastitis negative) of bacteria on culture, and the numbers of false negative and false positive results of the other three tests defined in relation to this. Similar numbers of false negative results were observed with the MCC (20), NAGase (18) and ELISA (13). False positive results due to the physiological factors present in early lactation were evident in the MCC, prominent in the NAGase test and absent from the ELISA. The major difference in false positive results associated with experimental infection between the three tests was the more rapid return to negative values of the ELISA following resolution of infection, compared with MCC and NAGase.

Enzyme histochemistry on muscle biopsies as an aid in the diagnosis of diseases of the equine neuromuscular system: a study of six cases.

Muscle biopsies from six horses with clinical histories of muscle atrophy, muscle tremors, myopathic symptoms, unsteadiness of pelvic limbs and progressive ataxia were examined. Muscle biopsies were studied with enzyme histochemical techniques to evaluate the diagnostic values of these methods in cases suspected of suffering from neuromuscular disorders. Hypertrophy, atrophy, fibre splitting, waxy degeneration, phagocytosis and necrosis were seen in haematoxylin eosin stained sections of the different cases. Fibre type predominance and fibre type grouping were seen in the calcium ion stimulated myosine ATP-ase (Ca-ATP-ase) stained sections of some cases. 'Moth-eaten fibres' were demonstrated in three cases by staining with NADH: nitro blue tetrazolium oxidoreductase (NADH-TR), succinate dehydrogenase (SDH), NADH dependent malate dehydrogenase, cytochrome c oxidase and by lactate dehydrogenase. The catabolic enzymes, acid phosphatase (ACP) and 5'-nucleotidase were active in cases with fibre phagocytosis. The oxidative part of the pentose phosphate pathway in myopathic tissue seemed to be important in three cases, demonstrated by the increased activity of glucose-6-phosphate dehydrogenase (GPDH) and 6-phosphogluconate dehydrogenase (PGDH). The important feature of diseased horse muscle was that the pathohistochemical changes were exactly the same as in diseased skeletal muscles of humans. The application of tissue saving enzyme histochemical techniques can be recommended in the study of muscle tissue from horses suffering from suspected neuromuscular disorders.

A three-step in vitro procedure for estimating intestinal digestion of protein in ruminants.

A three-step in vitro procedure was developed to estimate intestinal digestion of proteins in ruminants. Dacron bags containing feed samples were suspended in the rumen for 16 h. Residue containing 15 mg of N after ruminal exposure was incubated for 1 h in 10 mL of a .1 N HCl solution containing 1 g/L of pepsin. After incubation, pH was neutralized with .5 mL of 1 N NaOH and 13.5 mL of a pH 7.8 phosphate buffer containing 37.5 mg of pancreatin were added to the solution and incubated at 38 degrees C. After a 24-h incubation, 3 mL of a 100% (wt/vol) trichloroacetic acid solution were added to precipitate undigested proteins. Preincubation of samples in the rumen did not affect (P > .05) pepsin-pancreatin digestion of residual CP in soybean meal (SBM), corn gluten meal (CGM), and blood meal (BM) and reduced (P < .05) pepsin-pancreatin digestion of residual CP in hydrolyzed feather meal (HFM), fish meal (FM), and meat and bone meal (MBM) (80 vs 70, 88 vs 81, and 82 vs 56%, respectively, for nonruminal vs ruminal preincubation). Pepsin digestion before pancreatin digestion increased (P < .05) CP digestion of all proteins tested by a mean of 23 percentage units. The pancreatin digestion step was validated using 34 duodenal samples from which small intestinal CP digestion was determined in vivo. The regression equation of in vivo estimates on pancreatin digestion had an r value of .91 (P < .001).(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of a commercial creatine kinase screening test for malignant hyperthermia (porcine stress syndrome).

Two hundred and seven boars entering a Record of Performance Test Station at New Hamburg, Ontario were screened for susceptibility to malignant hyperthermia or the porcine stress syndrome. Screening tests included the determination of whole blood creatine kinase levels by a commercially available test using the method of bioluminescence and a halothane challenge. The validity of the bioluminescent or whole blood creatine kinase test as a screening mechanism for malignant hyperthermia was evaluated in boars in a field trial. The susceptibility of these animals to malignant hyperthermia or the porcine stress syndrome was unknown at the time of the study. It was determined in the initial field trial that 76.3% or 158 of the 207 boars tested by the bioluminescent creatine kinase screening test were stress susceptible. In contrast, only one boar (0.5%) reacted to a standard five minute halothane challenge. After further examination of the commercial screening test, little correlation was found to exist between the bioluminescent and Rosalki methods of creatine kinase determination. The coefficient of analytical variation for the bioluminescent method of creatine kinase determination ranged from 17.6% at a mean of 359 LU to 21.9% at a mean of 318 LU. Similarly, the coefficient for the Rosalki technique ranged from 7.1% at a mean of 324 U/L to 14.0% at a mean of 64 U/L. In addition, little correlation was found to exist between creatine kinase levels as determined by the bioluminescent method and outcome to the halothane or halothane/succinylcholine challenge, age of boars in weeks or percentage gain in weight.(ABSTRACT TRUNCATED AT 250 WORDS)

Diagnostic value of alkaline phosphatase isoenzyme separation by affinity electrophoresis in the dog.

Affinity electrophoresis, using wheat germ lectin, was used to separate the alkaline phosphatase isoenzymes in the sera of 150 dogs with alkaline phosphatase values greater than or equal to 150 IU/L. The method provided clearer separation of the liver, bone and steroid-induced alkaline phosphatase isoenzymes commonly observed in canine serum, compared to conventional cellulose acetate electrophoresis. The dogs were divided into four patient groups determined by previous corticosteroid treatment, evidence of elevated endogenous corticosteroid levels, age and alanine aminotransferase values. The isoenzyme pattern of each patient was qualitatively assessed. The isoenzyme pattern most frequently observed was greater than 50% steroid induced alkaline phosphatase, which was present in 76 of 150 dogs. This pattern was observed in 18 of 22 dogs receiving corticosteroid therapy, two of three dogs with hyperadrenocorticism, and in dogs with a variety of other diagnoses. The majority of immature dogs (12 of 20) had an isoenzyme pattern consisting of greater than 50% bone. The majority of dogs with active hepatocellular injury (16 of 27) had greater than 50% liver isoenzyme. The isoenzyme pattern was not specific for certain diseases, therefore the diagnostic usefulness is limited. However the isoenzyme result is useful in some cases to determine which further diagnostic tests are indicated, and to determine the source of alkaline phosphatase elevation.

Evaluation of urinary enzymes in dogs with early renal disorder.

We investigated urinary N-acetyl-beta-D-glucosaminidase NAG (EC 3.2.1.30), gamma-glutamyl transpeptidase gamma-GTP (EC 2.3.2.2) and glycyl-prolyl dipeptidyl aminopeptidase GP-DAP (EC 3.4.14.5) in dogs with heartworm disease and renal failure. In the renal failure dogs, the NAG, gamma-GTP and GP-DAP index were significantly higher than those in the healthy dogs. In the heartworm disease dogs with normal chest X-rays (HW I), none of the enzyme values was significantly different from those of the healthy controls. In the dogs with heartworm disease showing abnormal heart shadows on their chest X-rays (HW II), enzyme values were significantly higher than those in the healthy dogs (P < 0.01) and the HW I dogs (P < 0.01). Thus, these urinary enzymes tests are available for the early detection of renal disorders.

Diagnosis of Fasciola hepatica infection in beef calves by plasma enzyme analysis.

Plasma analysis for albumin, total bilirubin, and total protein values and aspartate aminotransferase (AST), arginase, and gamma-glutamyl transpeptidase (GGT) activities was used for the early and quantitative diagnosis of experimental Fasciola hepatica infections in beef calves. Calves were infected on 3 occasions with 1,000 (n = 5), 100 (n = 5), or 10 (n = 4) metacercariae for a total infective dose of 3,000, 300, or 30, respectively. Albendazole (15 mg/kg of body weight) was administered to 7 infected calves on postinfection (initial) week (PIW) 13. All calves were euthanatized and necropsied on PIW 16 for the determination of fluke infections. Plasma constituents were determined weekly. Significant (P less than 0.05) increases in AST activity occurred as early as PIW 4 and GGT activity at PIW 9, as compared with that in noninfected controls. Fluke burden-related differences were observed in GGT activity from PIW 9 onward. Increases in AST activity reflected parenchymal liver damage, whereas increases in GGT reflected hepatobiliary damage; therefore, differentiation could be made between the migratory and ductal phases of the infection. There was no correlation between arginase activity and fluke infection. As compared with fecal examination results, plasma enzyme analysis gave an earlier and semiquantitative indication of F hepatica infection in experimentally infected calves. Although increases in these plasma constituents were not definitely diagnostic of fascioliasis, useful information on the size of the fluke burden and progress of the disease process could be obtained by these methods. Plasma enzyme analyses of AST and GGT were not indicative of chemotherapeutic success or failure when calves with mature F hepatica (14 weeks old) infections were treated.

Determination of carbonic anhydrase III isoenzyme concentration in sera of racehorses with exertional rhabdomyolysis.

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.

Variations in serum sorbitol dehydrogenase, aspartate transaminase, and isoenzyme 5 of lactate dehydrogenase activities in horses given carbon tetrachloride.

Seven horses were given 0.5 mg of carbon tetrachloride/kg of body weight via a nasogastric tube. Subsequent hepatocellular damage was monitored by serum enzyme determinations of sorbitol dehydrogenase, isoenzyme 5 of lactate dehydrogenase, and aspartate transaminase activities. Creatinine kinase activity was evaluated as an indicator of muscle cell damage. Sorbitol dehydrogenase, isoenzyme 5 of lactate dehydrogenase, and aspartate transaminase activities were significantly (P less than 0.05) increased by 24 hours after carbon tetrachloride administration. Isoenzyme 5 of lactate dehydrogenase and sorbitol dehydrogenase activities returned to baseline several days before aspartate transaminase activity returned to baseline. Creatine kinase activity remained unchanged.

Circulating concentrations of trypsin-like immunoreactivity and activities of lipase and amylase after pancreatic duct ligation in dogs.

The possibility that assay of circulating trypsin-like immunoreactivity (TLI) could assist in the diagnosis of acute pancreatitis in dogs has been examined by assaying plasma TLI concentrations after pancreatic duct ligation and comparing the results with plasma activities of lipase and amylase. Venous blood samples were obtained from 8 dogs before surgery, then daily for 5 days and at 14 days after ligation of pancreatic ducts. Plasma concentrations of TLI increased within 24 hours and tended to peak before and to decrease more rapidly than activities of lipase and amylase, remaining greater than the control range for 5 days in all but 2 dogs. Plasma lipase and amylase activities increased together and remained greater than the control range in all dogs for 5 days after surgery. Regression analysis of all postoperative data indicated significant correlations between concentration of TLI and lipase activity (r = 0.67, P less than 0.001), concentration of TLI and amylase activity (r = 0.53, P less than 0.001), and between lipase and amylase activities (r = 0.74, P less than 0.001). These findings suggested that assay of TLI may provide an early indication of acute pancreatitis in dogs. Because TLI is specifically pancreatic in origin, high plasma TLI concentration may prove a more reliable indicator of clinical pancreatitis than high activities of amylase or lipase, which may be derived from extrapancreatic tissues.